Runx2 isoforms in angiogenesis

ABSTRACT

The present invention relates to RUNX2 and RUNXdelta8, and their use in modulating conditions and diseases associated with angiogenesis and cell proliferation. For example, RUNX2delta8 can be utilized to inhibit tumor growth and to prevent or inhibit angiogenesis. The present invention also relates to antibodies which specifically recognize RUNX2delta8, and distinguish it from RUNX2.

This application claims the benefit of U.S. Provisional Application Ser.No. 60/564,979, filed Apr. 26, 2004, which is hereby incorporated byreference in its entirety.

STATEMENT REGARDING GOVERNMENT RIGHTS

This invention was made with government support under NIH Grant Nos.CA95350 awarded by the National Institute of Health. The government hascertain rights in the invention.

BACKGROUND OF THE INVENTION

Neovascularization is an essential developmental process that isactivated under pathological conditions and controlled by the expressionof growth-promoting and growth-suppressing angiogenesis factors (Beck &D'Amore, 1997; Ferrara, 2000; Folkman, 1995; Kerbel, 2000; Li, 2000).During the initial activation stage, tumor or epithelial cells secreteangiogenic factors such as VEGF, FGF, and angiopoietins that alter cellcycle kinetics and stimulate EC proliferation or migration (Carmeliet &Collen, 2000; Hanahan, 1997; Maisonpierre et al., 1997). In laterstages, tube formation and vessel maturation lead to vessel remodelingand apoptosis, which are regulated by TGFβ₁ and mesenchymal cells in theabsence of EC proliferation (Beck & D'Amore, 1997; Pepper et al., 1990;Taipale & Keski-Oja, 1997). This complex process of gene expression isregulated by a variety of transcription factors whose functions inangiogenesis have been deduced from targeted gene disruption studies invivo or in cultured cells (Sato, 2000).

The Runx genes are a conserved family of DNA binding proteins containinga unique Runt homology domain (RHD) originally described in Drosophila(Ito, 1999). The RUNX proteins are members of the Ig-loop DNA bindingfamily of proteins that include Stat1, p53, and NFkB (Bravo et al.,2001). Runx proteins are phosphorylated (Selvamurugan et al., 2000; Xiaoet al., 2000) and associate with the core-binding factor-β (Cbfβ) in thenucleus to bind a specific nucleotide sequence. Several key observationssupport a role for Runx genes in angiogenesis including the finding thatmice in which either the Runx1 or Runx2 genes have been disrupted die inutero or soon after birth with vascular abnormalities (Li et al., 2002;Lund & Van Lohuizen, 2002). Runx1 negative mice fail to recruithematopoiefic stem cells for angiogenesis and exhibit defective vesselformation in the pericardium and head (Takakura et al., 2000). InRunx2-deficient mice, there is no vascular or mesenchymal cell invasionin cartilage, no evidence of VEGF expression in hypertrophicchondrocytes, and consequently no bone formation (Komori et al., 1997;Otto et al., 1997; Zelzer et al., 2001). The absence of VEGF may be adirect consequence of reduced Runx2 binding to the VEGF promoter (Zelzeret al., 2001). Conversely, VEGF, along with several angiogenic factorsincluding FGF-1 and IGF-1, stimulate RUNX2 expression and migration ofEC in vitro and in vivo (Namba et al., 2000; Sun et al., 2001). Reportsof RUNX1 expression in human vascular EC and brain tumor cells in vivoalso indicate that Runx genes are upregulated in highly-vascularizedmalignant tumors (Perry et al., 2002).

RUNX2 contains two domains not shared by other Runx family proteins: aQA-rich domain important in regulating transcription and a domain ofunknown function encoded in exon 8 (Westendorf & Hiebert, 1999).Alternatively-spliced Runx2 isoforms have been reported (Stewart et al.,1997), including alternatively spliced exon 8 (Zhang et al., 1997),which exhibited reduced transactivation relative to RUNX2 (Geoffroy aal., 1998), as well as isoforms arising from alternative transcriptionalstart sites (Xiao et al., 1998).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 (A-B). Expression of Runx2 in the rat aortic ring vascularsprouting assay. (A) Freshly dissected rat aorta were sectioned andcultured within clotted fibrin gels. Shown are aortic vessels (a) at thebeginning of the incubation, time=0 and 7 days later. Arrows indicatevascular sprouts. Bar (1 cm)=200 um. (B) Runx2, urokinase (uPA), VEGF,and membrane-type metalloproteinase (MT1MMP) expression. Total RNA wasextracted from fresh aortic vessels (T=0, lane 1) or vessels incubatedin fibrin gel for 7 days (T=7, lane 3) and RT-PCR was performed withgene-specific primers. Cyclophilin expression was used to control forgel loading and the presence of RNA. For controls, fibrin gel (FG)adjacent to vascular sprouts was extracted under identical conditions(FG, lane 2; no cyclophilin RNA detectable). Data are representative of3 separate experiments.

FIG. 2 (A-B). RUNX2 expression and mRNA stability in human EC. (A)Reduced RUNX2 levels in the absence of serum. RNA was isolated asdescribed in the Methods section and expression determined with specificprimers. Cyclophilin (CP) was used to normalize the RT-PCR reactions.HBME cells were grown to subconfluence, incubated in the absence of FBSfor 0 to 16 hours (lanes 1-6) and RNA prepared as described in theMethods. RT-PCR was performed using RUNX2-specific primers. Bandintensities were calculated by densitometry normalized to cyclophilincontrol and show a t_(1/2)=3 hr (A, right panel). Data arerepresentative of 3 separate experiments. (B) HBME cells that wereincubated in the absence of FBS for 16 hours were harvested withtrypsin/EDTA and 5×10⁵ cells/well were transferred to uncoated wells ofa 6-well plate (PL; lanes 1,2), wells coated with EHS matrix gel (MG;lanes 3,4) or wells coated with fibrin gel (FG; lanes 5,6) in thepresence of 10% FBS (lanes 1,3,5) or 0.1% BSA (lanes 2,4,6). Cells wereincubated for 6 hours and RNA was prepared for RT-PCR withRUNX2-specific primers.

FIG. 3 (A-B). Nucleotide sequence of a natural, alternative splicevariant and RUNX2 functional domains. (A) Sequence of RUNX2 isoformsfrom HBME cells. Shown are the sequences for the 416 bp (RUNX2) (SEQ IDNOS 40-41, respectively, in order or appearance) and 350 by (RUNX2Δ8)(SEQ ID NOS 38-39, respectively, in order or appearance) PCR products.Sequence analysis of these PCR products showed that both were containedwithin human RUNX2 with the 416 bp band encompassing exons 6, 7, and 8while the 350 bp band was the result of an exact 66 bp deletion of exon8. The PCR primers used are shown in large type and the exon 8nucleotides are underlined with the exon boundaries shown in verticallines. Also shown are the amino acid sequences for RUNX2 and RUNX2Δ8with the exon 8 peptide underlined. The N-terminal Asp residue withinexon 8 is encoded by GAT while the C-terminal Ala residue is encoded byGCA. (B) Exon and functional map of RUNX2. Shown are the relativelocations of the QA activation domain (AD2), the Runt DNA bindingdomain, the NLS nuclear localization signal, activation domain 3 (AD3)and the transcriptional repression domains RD and WRPY (SEQ ID NO: 45).The location of exon 8 within the RUNX2 functional domains and thepredicted amino acid sequence (SEQ ID NO: 42) are shown.

FIG. 4 (A-C). RUNX2 DNA-binding activity and EC proliferation. (A)Gel-shift assay to detect RUNX2 DNA-binding activity. Nuclear extractsprepared from HBME cells, which express endogenous RUNX2, were incubatedwithout (lane 1) or with (lanes 2-5) increasing amounts ofRUNX2-specific antibody and with the consensus RUNX2-bindingoligonucleotide (SEQ ID NOS 43-44, respectively, in order or appearance)to verify the presence of RUNX2 in the binding complex. (B) RUNX2DNA-binding activity in quiescent and proliferative EC. Nuclear extractsfrom subconfluent (lanes 1-4) or postconfluent (lanes 5-8) HBME cellswere incubated with Runx2 antibody (lanes 2,6), with 100-fold excessunlabeled specific RUNX2-binding oligos (lanes 3,7), with 100-foldexcess non-specific STAT-binding oligos (lanes 4,8), or were leftuntreated (lanes 1,5). (C) RUNX2 DNA-binding activity was determined inserum-starved HBME cells (lane 1), serum-starved HBME treated with 20ng/ml IGF-1 (lane 2), or HEK293 cells transfected with Flag.tag RUNX2(lane 3), or Flag.tag RUNX2Δ8 expressing plasmid (lane 4). Panel arepresents EMSA, panel b represents Western blot of nuclear extracts forendogenous RUNX2 and RUNX2Δ8 (lanes 1,2) or Flag.tag RUNX2 or RUNX2Δ8(lanes 3,4), and panel c is the Western blot loading control (totalAkt). All samples were resolved on TBE-acrylamide DNA retardation gels.Arrows (A,B) indicate the RUNX2-specific shifted complex (b) and theRUNX2 antibody super-shifted complex (a). Similar results were obtainedin three additional experiments.

FIG. 5 (A-D). Ectopic expression of RUNX2 isoforms and EC proliferation.(A) Protein expression in HEK293, HBME, or BAEC cells transfected withFlag-tagged vectors encoding RUNX2 isoforms was detected by Westernblots using M2 (Flag-tag; for HBME and BAEC) or AML3 (RUNX2; for HBME)antibodies. The blots were re-probed with γ-tubulin specific antibodiesto verify equal loading. Flag-tag proteins from transfected BAEC wereimmunoprecipitated with M2 antibody and subjected to Western blotting.Ig-LC=immunoglobulin light chain. Data for BAEC are representative of 4different sets of stable, polyclonal transfectants. (B) Stabletransfectants of BAEC (Neo, RUNX2 and RUNX2Δ8) were grown to confluence,harvested, and re-cultured in 96-well plates (1×10⁴ cells/well). Cellswere incubated for 4, 24, 48 or 72 hours at 37° C. MTT dye was used todetect viable cells. Absorbance at 540 nm was measured and expressed asthe mean and SD from n=4-6 per point. (Bars=SD; *p<0.01 versus RUNX2Δ8or neo at 72 hr). (C) Stable transfectants of BAEC (Neo, RUNX2 andRUNX2Δ8) were re-plated after confluence in a 96-well plate inserum-containing medium for 4 h, 8 h, 24 h, 48 h or 72 h, with(³H)-thymidine added to the medium for the final 1 hour. Total cellularprotein was determined from duplicate cultures and incorporation oflabel was normalized to cell protein content. n=3 for each experimentalgroup. (p<0.01 versus RUNX2Δ8 or neo at 24 or 48 hr). (D) BAEC cellswere transfected with 0.6 ug of the indicated cDNA and 6 ul lipofectinfor 4 hr in serum-free media. Serum (10%) was added for 18 hr and thecells were serum starved for 48 hr prior to treatment with 10% FBS for0, 3, or 6 hr as indicated. Western blotting with anti-Rb and γ-tubulincontrol antibodies is shown.

FIG. 6 (A-C). RUNX2 suppresses the TGFβ₁-mediated inhibition of ECproliferation. (A) BAEC transfectants were cultured in 6-well plates(5×10⁵/well) and treated with TGFβ₁ (0, 0.2, 2.0, 20 ng/ml) for 48hours. Cells were fixed with PBS-buffered formalin and stained with DAPIto visualize nuclei at 320× magnification. (B) For each treatmentcondition, cells in the fluorescent images were counted (fourfields/well) and expressed as the number of cells/field versus TGFβ₁concentration. The data represent the mean and standard deviations(bars) with * indicating statistically significant differences betweenRUNX2 and neo or RUNX2Δ8 transfectants (p<0.01 at 0.2 ng/ml; p<0.05 at2.0 and 20 ng/ml). (C) BAEC transfected with control (NEO), RUNX2, orRUNX2Δ8 were spot cultured in 6-well plates and overlayered withcollagen gel (3 mg/ml) in the presence of TGFβ (10 ng/ml) for 48 hr(left panels). Higher magnification shows a representative cell fromeach well indicating DNA fragmentation in RUNX2Δ8 transfectants (rightpanel).

FIG. 7 (A-E). RUNX2Δ8 reduces survival in TGFβ₁-treated EC. (A,B) BAEC(5×10⁵/well) were cultured in 6-well plates in the presence of 0.2% FBSand TGFβ₁ (2 ng/ml) for 28 hours. Cells were then fixed withPBS-buffered formalin, stained with DAPI to detect nuclei andphotographed with an epifluorescence microscope. Neo, RUNX2, and RUNX2Δ8transfectants were compared under fluorescence (A) or phase contrast(B). (Bars, 1 cm, =100 um). (C) The degree of apoptosis is expressed asthe % of total cells in a given photographic field whose nuclei displaythe morphologic features of apoptosis. At least 3 photographic fieldsfor each transfectant were used for quantitation. (D) Detached,DAPI-stained cell nuclei were counted under low power and the number ofdetached cells per field (a minimum of 3 fields per transfectant) werequantitated. All detached cells exhibited condensed nucleicharacteristic of apoptosis. Statistical significance was calculatedusing Student's t-test from the means±SD. (*p<0.01 versus neo or RUNX2)(E) Representative Western blot of neo (lane 1), RUNX2 (lane 2), andRUNX2Δ8 (lane 3) transfected EC after TGFβ₁ treatment. Anti-Parpantibodies were used to confirm the presence of caspase activity. Arrowsindicate uncleaved and cleaved Parp substrate.

FIG. 8 (A-D). Regulation of the p21^(CIP1) promoter by RUNX2 andRUNX2Δ8. (A) RUNX2 and RUNX2Δ8 binding to a p21^(CIP1) promoterRUNX-binding element. HEK293 cells were transfected with Flag.tag RUNX2(lane 1), RUNX2Δ8 (lane 2), or control (lane 3) vectors, nuclearextracts were prepared, and Flag.tag proteins were detected by Westernblotting. Nuclear extracts were incubated with biotin-labeled wild-type(lanes 4,6) or mutant (lanes 5,7) double-stranded oligonucleotides, theprotein-DNA complexes were isolated with Streptavidin beads, and theFlag.tag RUNX2 (lanes 4,5) or RUNX2Δ8 (lanes 6,7) proteins were detectedby Western blotting. The lower panel indicates the ability of RUNX2Δ8(Δ8) to compete with RUNX2 (R2) for the DNA binding site. Increasingamounts of nuclear protein from RUNX2Δ8-transfected cells were incubatedwith 500 ug of protein from RUNX2-transfected cells and RUNX2 or RUNX2Δ8bound to DNA was detected by Western blotting. Arrows indicate positionsof each isoform. Figure discloses SEQ ID NOS 19-22, respectively, inorder or appearance (B,C,D) NIH3T3 cells (2×10⁵/well) were transfectedwith the Mirus LT1 reagent and the p21^(CIP1)-promoter luciferaseplasmid in the presence of TK-Renilla plasmid as control. The neo,RUNX2, or RUNX2Δ8 plasmids were co-transfected for 42 hours and cellswere left untreated (B) or treated (C) with TGFβ₁ (2 ng/ml) for anadditional 6 h prior to preparation of the lysates for analysis with theDual-Luciferase system. For competition experiments (D), basal levels ofRUNX2 (0.25 ug), Alk5TD (0.05 ug), and Smad3 (0.05 ug) plasmids wereco-transfected with RUNX2Δ8 plasmid (0.05 ug) as indicated. Eachtransfection was performed in triplicate and measurements were recordedtwo separate times. For detection of p21^(CIP1) protein (D, inset),cells were treated with 0.1 uM doxorubicin for 24 hr prior to analysisof nuclear extracts by Western blotting. Each experiment was repeatedfour (B) or three (C,D) times. Firefly luciferase activity relative toRenilla luciferase (B, D) or the fold-change in repression or activation(C) was calculated relative to untransfected cells. Mean and standarddeviation are shown. Statistical significance between RUNX2 and NEOtransfected cells (p<0.05) is indicated by the asterisk.

FIG. 9 (A-D) shows amino acid sequences of RUNX polypeptides. RUNX1 isSEQ ID NO: 1, RUNX2 is SEQ ID NO: 35; RUNX2delta8 is SEQ ID NO: 3; andRUNX3 is SEQ ID NO:2.

FIG. 10 (A and B) shows the nucleotide (SEQ ID NO: 34) and amino acidsequence (SEQ ID NO: 35) of RUNX2. The amino acid and nucleotidesequences of exon 8 are underlined. The nucleotide sequence ofRUNXdelta8 contains nucleotide positions 1-979 and 1045-1486 (i.e.,where the coding sequence for exon 8 are deleted). See, NCBI Accessionnumbers NM_(—)004348 and NP_(—)004339.

FIG. 11 (and B) show the nucleotide (SEQ ID NO: 36) and amino acidsequence (SEQ ID NO: 37) of YAP (yes-associated protein). NCBI AccessionNo. X80507.

DESCRIPTION OF THE INVENTION

The present invention relates to all facets of the RUNX, including theRUNX2delta8 isoform, polynucleotides thereof, polypeptides encoded bythem, antibodies and specific binding partners thereto, and theirapplications to research, diagnosis, drug discovery, therapy, clinicalmedicine, forensic science and medicine, etc. The polynucleotides,polypeptides, and antibodies are useful in variety of ways, including,but not limited to, as molecular markers for angiogenesis, as drugtargets, and for detecting, diagnosing, staging, monitoring,prognosticating, preventing or treating, determining predisposition to,etc., diseases and conditions relating to angiogenesis, cellproliferation, and cell cycle control of endothelial and tumor cells.Agents of the present invention can be used to regulate cancer, heartdisease, stroke, diabetic retinopathy, and macular degeneration.

Nucleic Acids

A mammalian polynucleotide, or fragment thereof, of the presentinvention is a polynucleotide having a nucleotide sequence obtainablefrom a natural source. When the species name is used, e.g., humanRUNX2deltaA8, it indicates that the polynucleotide or polypeptide isobtainable from a natural source. It therefore includesnaturally-occurring normal, naturally-occurring mutant, andnaturally-occurring polymorphic alleles (e.g., SNPs),differentially-spliced transcripts, splice-variants, etc. By the term“naturally-occurring,” it is meant that the polynucleotide is obtainablefrom a natural source, e.g., animal tissue and cells, body fluids,tissue culture cells, forensic samples. Natural sources include, e.g.,living cells obtained from tissues and whole organisms, tumors, culturedcell lines, including primary and immortalized cell lines.

Naturally-occurring mutations can include deletions (e.g., a truncatedamino- or carboxy-terminus), substitutions, inversions, or additions ofnucleotide sequence. These genes can be detected and isolated bypolynucleotide hybridization according to methods which one skilled inthe art would know, e.g., as discussed below.

A polynucleotide according to the present invention can be obtained froma variety of different sources. It can be obtained from DNA or RNA, suchas polyadenylated mRNA or total RNA, e.g., isolated from tissues, cells,or whole organism. The polynucleotide can be obtained directly from DNAor RNA, from a cDNA library, from a genomic library, etc. Thepolynucleotide can be obtained from a cell or tissue (e.g., from anembryonic or adult tissues) at a particular stage of development, havinga desired genotype, phenotype, disease status, etc. A polynucleotidewhich “codes without interruption” refers to a polynucleotide having acontinuous open reading frame (“ORF”) as compared to an ORF which isinterrupted by introns or other noncoding sequences.

Polynucleotides and polypeptides (including any part of RUNX2 DBLTA8)can be excluded as compositions from the present invention if, e.g.,listed in a publicly available databases on the day this application wasfiled and/or disclosed in a patent application having an earlier filingor priority date than this application and/or conceived and/or reducedto practice earlier than a polynucleotide in this application.

Specific Polynucleotide Probes

A polynucleotide of the present invention can comprise any continuousnucleotide sequence of SEQ ID NO:3, sequences which share sequenceidentity thereto, or complements thereof. The term “probe” refers to anysubstance that can be used to detect, identify, isolate, etc., anothersubstance. A polynucleotide probe is comprised of nucleic acid can beused to detect, identify, etc., other nucleic acids, such as DNA andRNA.

These polynucleotides can be of any desired size that is effective toachieve the specificity desired. For example, a probe can be from about7 or 8 nucleotides to several thousand nucleotides, depending upon itsuse and purpose. For instance, a probe used as a primer PCR can beshorter than a probe used in an ordered array of polynucleotide probes.Probe sizes vary, and the invention is not limited in any way by theirsize, e.g., probes can be from about 7-2000 nucleotides, 7-1000, 8-700,8-600, 8-500, 8-400, 8-300, 8-150, 8-100, 8-75, 7-50, 10-25, 14-16, atleast about 8, at least about 10, at least about 15, at least about 25,etc. The polynucleotides can have non-naturally-occurring nucleotides,e.g., inosine, AZT, 3TC, etc. The polynucleotides can have 100% sequenceidentity or complementarity to a sequence of SEQ ID NO:3, or it can havemismatches or nucleotide substitutions, e.g., 1, 2, 3, 4, or 5substitutions.

In accordance with the present invention, a polynucleotide can bepresent in a kit, where the kit includes, e.g., one or morepolynucleotides, a desired buffer (e.g., phosphate, tris, etc.),detection compositions, RNA or cDNA from different tissues to be used ascontrols, libraries, etc. The polynucleotide can be labeled orunlabeled, with radioactive or non-radioactive labels as known in theart. Kits can comprise one or more pairs of polynucleotides foramplifying nucleic acids specific for RUNX2DELTAA8. These include bothsense and anti-sense orientations. For instance, in PCR-based methods(such as RT-PCR), a pair of primers are typically used, one having asense sequence and the other having an antisense sequence.

Another aspect of the present invention is a nucleotide sequence that isspecific to, or for, a selective polynucleotide. The phrases “specificfor” or “specific to” a polynucleotide have a functional meaning thatthe polynucleotide can be used to identify the presence of one or moretarget genes in a sample and distinguish them from non-target genes. Itis specific in the sense that it can be used to detect polynucleotidesabove background noise (“non-specific binding”). A specific sequence isa defined order of nucleotides (or amino acid sequences, if it is apolypeptide sequence) which occurs in the polynucleotide, e.g., in thenucleotide sequences of SEQ ID NO:3, and which is characteristic of thattarget sequence, and substantially no non-target sequences. A probe ormixture of probes can comprise a sequence or sequences that are specificto a plurality of target sequences, e.g., where the sequence is aconsensus sequence, a functional domain, etc., e.g., capable ofrecognizing a family of related genes. Such sequences can be used asprobes in any of the methods described herein or incorporated byreference. Both sense and antisense nucleotide sequences are included. Aspecific polynucleotide according to the present invention can bedetermined routinely.

A polynucleotide comprising a specific sequence can be used as ahybridization probe to identify the presence of, e.g., humanpolynucleotide, in a sample comprising a mixture of polynucleotides,e.g., on a Northern blot. Hybridization can be performed under highstringent conditions (see, above) to select polynucleotides (and theircomplements which can contain the coding sequence) having at least 90%,95%, 99%, etc., identity (i.e., complementarity) to the probe, but lessstringent conditions can also be used. A specific polynucleotidesequence can also be fused in-frame, at either its 5′ or 3′ end, tovarious nucleotide sequences as mentioned throughout the patent,including coding sequences for enzymes, detectable markers, GFP, etc,expression control sequences, etc.

A polynucleotide probe, especially one that is specific to apolynucleotide of the present invention, can be used in gene detectionand hybridization methods as already described. In one embodiment, aspecific polynucleotide probe can be used to detect whether a particulartissue or cell-type is present in a target sample. To carry out such amethod, a selective polynucleotide can be chosen which is characteristicof the desired target tissue. Such polynucleotide is preferably chosenso that it is expressed or displayed in the target tissue, but not inother tissues which are present in the sample. For instance, ifdetection of endothelial cells is desired, it may not matter whether theselective polynucleotide is expressed in other tissues. Starting fromthe selective polynucleotide, a specific polynucleotide probe can bedesigned which hybridizes (if hybridization is the basis of the assay)under the hybridization conditions to the selective polynucleotide,whereby the presence of the selective polynucleotide can be determined.

Probes which are specific for polynucleotides of the present inventioncan also be prepared using involve transcription-based systems, e.g.,incorporating an RNA polymerase promoter into a selective polynucleotideof the present invention, and then transcribing anti-sense RNA using thepolynucleotide as a template. See, e.g., U.S. Pat. No. 5,545,522.

Nucleic Acid Detection Methods

Another aspect of the present invention relates to methods and processesfor detecting RUNX2deltaA8. Detection methods have a variety ofapplications, including for diagnostic, prognostic, forensic, andresearch applications. To accomplish gene detection, a polynucleotide inaccordance with the present invention can be used as a “probe.” The term“probe” or “polynucleotide probe” has its customary meaning in the art,e.g., a polynucleotide which is effective to identify (e.g., byhybridization), when used in an appropriate process, the presence of atarget polynucleotide to which it is designed. Identification caninvolve simply determining presence or absence, or it can bequantitative, e.g., in assessing amounts of a gene or gene transcriptpresent in a sample. Probes can be useful in a variety of ways, such asfor diagnostic purposes, to identify homologs, and to detect,quantitate, or isolate a polynucleotide of the present invention in atest sample.

Assays can be utilized which permit quantification and/orpresence/absence detection of a target nucleic acid in a sample. Assayscan be performed at the single-cell level, or in a sample comprisingmany cells, where the assay is “averaging” expression over the entirecollection of cells and tissue present in the sample. Any suitable assayformat can be used, including, but not limited to, e.g., Southern blotanalysis, Northern blot analysis, polymerase chain reaction (“PCR”)(e.g., Saiki et al., Science, 241:53, 1988; U.S. Pat. Nos. 4,683,195,4,683,202, and 6,040,166; PCR Protocols: A Guide to Methods andApplications, Innis et al., eds., Academic Press, New York, 1990),reverse transcriptase polymerase chain reaction (“RT-PCR”), anchoredPCR, rapid amplification of cDNA ends (“RACE”) (e.g., Schaefer in GeneCloning and Analysis: Current Innovations, Pages 99-115, 1997), ligasechain reaction (“LCR”) (EP 320 308), one-sided PCR (Ohara et al., Proc.Natl. Acad. Sci., 86:5673-5677, 1989), indexing methods (e.g., U.S. Pat.No. 5,508,169), in situ hybridization, differential display (e.g., Lianget al., Nucl. Acid. Res., 21:3269-3275, 1993; U.S. Pat. Nos. 5,262,311,5,599,672 and 5,965,409; WO97/18454; Prashar and Weissman, Proc. Natl.Acad. Sci., 93:659-663, and U.S. Pat. Nos. 6,010,850 and 5,712,126;Welsh et al., Nucleic Acid Res., 20:4965-4970, 1992, and U.S. Pat. No.5,487,985) and other RNA fingerprinting techniques, nucleic acidsequence based amplification (“NASBA”) and other transcription basedamplification systems (e.g., U.S. Pat. Nos. 5,409,818 and 5,554,527; WO88/10315), polynucleotide arrays (e.g., U.S. Pat. Nos. 5,143,854,5,424,186; 5,700,637, 5,874,219, and 6,054,270; PCT WO 92/10092; PCT WO90/15070), Qbeta Replicase (PCT/US87/00880), Strand DisplacementAmplification (“SDA”), Repair Chain Reaction (“RCR”), nucleaseprotection assays, subtraction-based methods, Rapid-Scan™, etc.Additional useful methods include, but are not limited to, e.g.,template-based amplification methods, competitive PCR (e.g., U.S. Pat.No. 5,747,251), redox-based assays (e.g., U.S. Pat. No. 5,871,918),Taqman-based assays (e.g., Holland et al., Proc. Natl. Acad, Sci.,88:7276-7280, 1991; U.S. Pat. Nos. 5,210,015 and 5,994,063), real-timefluorescence-based monitoring (e.g., U.S. Pat. No. 5,928,907), molecularenergy transfer labels (e.g., U.S. Pat. Nos. 5,348,853, 5,532,129,5,565,322, 6,030,787, and 6,117,635; Tyagi and Kramer, Nature Biotech.,14:303-309, 1996). Any method suitable for single cell analysis of geneor protein expression can be used, including in situ hybridization,immunocytochemistry, MACS, FACS, flow cytometry, etc. For single cellassays, expression products can be measured using antibodies, PCR, orother types of nucleic acid amplification (e.g., Brady et al., MethodsMol. & Cell. Biol. 2, 17-25, 1990; Eberwine et al., 1992, Proc. Natl.Acad. Sci., 89, 3010-3014, 1992; U.S. Pat. No. 5,723,290). These andother methods can be carried out conventionally, e.g., as described inthe mentioned publications.

Many of such methods may require that the polynucleotide is labeled, orcomprises a particular nucleotide type useful for detection. The presentinvention includes such modified polynucleotides that are necessary tocarry out such methods. Thus, polynucleotides can be DNA, RNA, DNA:RNAhybrids, PNA, etc., and can comprise any modification or substituentwhich is effective to achieve detection.

Detection can be desirable for a variety of different purposes,including research, diagnostic, prognostic, and forensic. For diagnosticpurposes, it may be desirable to identify the presence or quantity of apolynucleotide sequence in a sample, where the sample is obtained fromtissue, cells, body fluids, etc. In a preferred method as described inmore detail below, the present invention relates to a method ofdetecting a polynucleotide comprising, contacting a targetpolynucleotide in a test sample with a polynucleotide probe underconditions effective to achieve hybridization between the target andprobe; and detecting hybridization.

Any test sample in which it is desired to identify a polynucleotide orpolypeptide thereof can be used, including, e.g., blood, urine, saliva,stool (for extracting nucleic acid, see, e.g., U.S. Pat. No. 6,177,251),swabs comprising tissue, biopsied tissue, tissue sections, culturedcells, etc.

Detection can be accomplished in combination with polynucleotide probesfor other genes, e.g., genes which are expressed in other diseasestates, tissues, cells, such as brain, heart, kidney, spleen, thymus,liver, stomach, small intestine, colon, muscle, lung, testis, placenta,pituitary, thyroid, skin, adrenal gland, pancreas, salivary gland,uterus, ovary, prostate gland, peripheral blood cells (T-cells,lymphocytes, etc.), embryo, normal breast fat, adult and embryonic stemcells, specific cell-types, such as endothelial, epithelial, myocytes,adipose, luminal epithelial, basoepithelial, myoepithelial, stromalcells, etc.

Polynucleotides can be used in wide range of methods and compositions,including for detecting, diagnosing, staging, grading, assessing,prognosticating, etc. diseases and disorders associated withRUNX2delta8, for monitoring or assessing therapeutic and/or preventativemeasures, in ordered arrays, etc. Any method of detecting genes andpolynucleotides of SEQ ID NO:3 can be used; certainly, the presentinvention is not to be limited how such methods are implemented.

Along these lines, the present invention relates to methods of detectingRUNX2delta8 in a sample comprising nucleic acid. Such methods cancomprise one or more the following steps in any effective order, e.g.,contacting said sample with a polynucleotide probe under conditionseffective for said probe to hybridize specifically to nucleic acid insaid sample, and detecting the presence or absence of probe hybridizedto nucleic acid in said sample, wherein said probe is, e.g., apolynucleotide which is SEQ ID NO:3, a polynucleotide having, e.g.,about 70%, 80%, 85%, 90%, 95%, 99%, or more sequence identity thereto,effective or specific fragments thereof, or complements thereto. Thedetection method can be applied to any sample, e.g., cultured primary,secondary, or established cell lines, tissue biopsy, blood, urine,stool, cerebral spinal fluid, and other bodily fluids, for any purpose.

Contacting the sample with probe can be carried out by any effectivemeans in any effective environment. It can be accomplished in a solid,liquid, frozen, gaseous, amorphous, solidified, coagulated, colloid,etc., mixtures thereof, matrix. For instance, a probe in an aqueousmedium can be contacted with a sample which is also in an aqueousmedium, or which is affixed to a solid matrix, or vice-versa.

Generally, as used throughout the specification, the term “effectiveconditions” means, e.g., the particular milieu in which the desiredeffect is achieved. Such a milieu, includes, e.g., appropriate buffers,oxidizing agents, reducing agents, pH, co-factors, temperature, ionconcentrations, suitable age and/or stage of cell (such as, inparticular part of the cell cycle, or at a particular stage whereparticular genes are being expressed) where cells are being used,culture conditions (including substrate, oxygen, carbon dioxide, etc.).When hybridization is the chosen means of achieving detection, the probeand sample can be combined such that the resulting conditions arefunctional for said probe to hybridize specifically to nucleic acid insaid sample.

The phrase “hybridize specifically” indicates that the hybridizationbetween single-stranded polynucleotides is based on nucleotide sequencecomplementarity. The effective conditions are selected such that theprobe hybridizes to a preselected and/or definite target nucleic acid inthe sample. For instance, if detection of a polynucleotide set forth inSEQ ID NO:3 is desired, a probe can be selected which can hybridize tosuch target gene under high stringent conditions, without significanthybridization to other genes in the sample. To detect homologs of apolynucleotide set forth in SEQ ID NO:3, the effective hybridizationconditions can be less stringent, and/or the probe can comprise codondegeneracy, such that a homolog is detected in the sample.

As already mentioned, the methods can be carried out by any effectiveprocess, e.g., by Northern blot analysis, polymerase chain reaction(PCR), reverse transcriptase PCR, RACE PCR, in situ hybridization, etc.,as indicated above. When PCR based techniques are used, two or moreprobes are generally used. One probe can be specific for a definedsequence which is characteristic of a selective polynucleotide, but theother probe can be specific for the selective polynucleotide, orspecific for a more general sequence, e.g., a sequence such as polyAwhich is characteristic of mRNA, a sequence which is specific for apromoter, ribosome binding site, or other transcriptional features, aconsensus sequence (e.g., representing a functional domain). For theformer aspects, 5′ and 3′ probes (e.g., polyA, Kozak, etc.) arepreferred which are capable of specifically hybridizing to the ends oftranscripts. When PCR is utilized, the probes can also be referred to as“primers” in that they can prime a DNA polymerase reaction.

In addition to testing for the presence or absence of polynucleotides,the present invention also relates to determining the amounts at whichpolynucleotides of the present invention are expressed in sample anddetermining the differential expression of such polynucleotides insamples. Such methods can involve substantially the same steps asdescribed above for presence/absence detection, e.g., contacting withprobe, hybridizing, and detecting hybridized probe, but using morequantitative methods and/or comparisons to standards.

The amount of hybridization between the probe and target can bedetermined by any suitable methods, e.g., PCR, RT-PCR, RACE PCR,Northern blot, polynucleotide microarrays, Rapid-Scan, etc., andincludes both quantitative and qualitative measurements. For furtherdetails, see the hybridization methods described above and below.Determining by such hybridization whether the target is differentiallyexpressed (e.g., up-regulated or down-regulated) in the sample can alsobe accomplished by any effective means. For instance, the target'sexpression pattern in the sample can be compared to its pattern in aknown standard, such as in a normal tissue, or it can be compared toanother gene in the same sample. When a second sample is utilized forthe comparison, it can be a sample of normal tissue that is known not tocontain diseased cells. The comparison can be performed on samples whichcontain the same amount of RNA (such as polyadenylated RNA or totalRNA), or, on RNA extracted from the same amounts of starting tissue.Such a second sample can also be referred to as a control or standard.Hybridization can also be compared to a second target in the same tissuesample. Experiments can be performed that determine a ratio between thetarget nucleic acid and a second nucleic acid (a standard or control),e.g., in a normal tissue. When the ratio between the target and controlare substantially the same in a normal and sample, the sample isdetermined or diagnosed not to contain cells. However, if the ratio isdifferent between the normal and sample tissues, the sample isdetermined to contain endothelial cells. The approaches can be combined,and one or more second samples, or second targets can be used. Anysecond target nucleic acid can be used as a comparison, including“housekeeping” genes, such as beta-actin, alcohol dehydrogenase, or anyother gene whose expression does not vary depending upon the diseasestatus of the cell.

Polynucleotide Expression and Polypeptides Produced Thereby Thereto

A polynucleotide according to the present invention can be expressed ina variety of different systems, in vitro and in vivo, according to thedesired purpose. For example, a polynucleotide can be inserted into anexpression vector, introduced into a desired host, and cultured underconditions effective to achieve expression of a polypeptide coded for bythe polynucleotide, to search for specific binding partners. Effectiveconditions include any culture conditions which are suitable forachieving production of the polypeptide by the host cell, includingeffective temperatures, pH, medium, additives to the media in which thehost cell is cultured (e.g., additives which amplify or induceexpression such as butyrate, or methotrexate if the codingpolynucleotide is adjacent to a dhfr gene), cycloheximide, celldensities, culture dishes, etc. A polynucleotide can be introduced intothe cell by any effective method including, e.g., naked DNA, calciumphosphate precipitation, electroporation, injection, DEAE-Dextranmediated transfection, fusion with liposomes, association with agentswhich enhance its uptake into cells, viral transfection. A cell intowhich a polynucleotide of the present invention has been introduced is atransformed host cell. The polynucleotide can be extrachromosomal orintegrated into a chromosome(s) of the host cell. It can be stable ortransient. An expression vector is selected for its compatibility withthe host cell. Host cells include, mammalian cells, e.g., COS, CV1, BHK,CHO, HeLa, LTK, NIH 3T3, 293, endothelial, epithelial, muscle, embryonicand adult stem cells, ectodermal, mesenchymal, endodermal, neoplastic,blood, bovine CPAE (CCL-209), bovine FBHE (CRL-1395), humanHUV-EC-C(CRL-1730), mouse SVEC4-10EHR1 (CRL-2161), mouse MS1 (CRL-2279),mouse MS1 VEGF (CRL-2460), insect cells, such as Sf9 (S. frugipeda) andDrosophila, bacteria, such as E. coli, Streptococcus, bacillus, yeast,such as Sacharomyces, S. cerevisiae, fungal cells, plant cells,embryonic or adult stem cells (e.g., mammalian, such as mouse or human).

Expression control sequences are similarly selected for hostcompatibility and a desired purpose, e.g., high copy number, highamounts, induction, amplification, controlled expression. Othersequences which can be employed include enhancers such as from SV40,CMV, RSV, inducible promoters, cell-type specific elements, or sequenceswhich allow selective or specific cell expression. Promoters that can beused to drive its expression, include, e.g., the endogenous promoter,MMTV, SV40, trp, lac, tac, or T7 promoters for bacterial hosts; or alphafactor, alcohol oxidase, or PGH promoters for yeast. RNA promoters canbe used to produced RNA transcripts, such as T7 or SP6. See, e.g.,Melton et al., Polynucleotide Res., 12(18):7035-7056, 1984; Dunn andStudier. J. Mol. Bio., 166:477-435, 1984; U.S. Pat. No. 5,891,636;Studier et al., Gene Expression Technology, Methods in Enzymology,85:60-89, 1987. In addition, as discussed above, translational signals(including in-frame insertions) can be included.

When a polynucleotide is expressed as a heterologous gene in atransfected cell line, the gene is introduced into a cell as describedabove, under effective conditions in which the gene is expressed. Theterm “heterologous” means that the gene has been introduced into thecell line by the “hand-of-man.” Introduction of a gene into a cell lineis discussed above. The transfected (or transformed) cell expressing thegene can be lysed or the cell line can be used intact.

For expression and other purposes, a polynucleotide can contain codonsfound in a naturally-occurring gene, transcript, or cDNA, for example,e.g., as set forth in SEQ ID NO:3, or it can contain degenerate codonscoding for the same amino acid sequences. For instance, it may bedesirable to change the codons in the sequence to optimize the sequencefor expression in a desired host. See, e.g., U.S. Pat. Nos. 5,567,600and 5,567,862.

A polypeptide according to the present invention can be recovered fromnatural sources, transformed host cells (culture medium or cells)according to the usual methods, including, detergent extraction (e.g.,non-ionic detergent, Triton X-100, CHAPS, octylglucoside, IgepalCA-630), ammonium sulfate or ethanol precipitation, acid extraction,anion or cation exchange chromatography, phosphocellulosechromatography, hydrophobic interaction chromatography, hydroxyapatitechromatography, lectin chromatography, gel electrophoresis. Proteinrefolding steps can be used, as necessary, in completing theconfiguration of the mature protein. Finally, high performance liquidchromatography (HPLC) can be employed for purification steps. Anotherapproach is express the polypeptide recombinantly with an affinity tag(Flag epitope, HA epitope, myc epitope, 6×His (SEQ ID NO: 46), maltosebinding protein, chitinase, etc) and then purify by anti-tagantibody-conjugated affinity chromatography.

Antibodies and Specific-Binding Partners to RUNX

The present invention also relates to polypeptides of RUNX2delta8, e.g.,an isolated mammalian (e.g. human) RUNX2delta8 polypeptide comprising orhaving the amino acid sequence set forth in FIG. 3, an isolated humanRUNX2delta8 polypeptide comprising an amino acid sequence having 90%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more amino acid sequenceidentity to the amino acid sequence set forth in SEQ ID NO 2, andoptionally having one or more of RUNX2delta8 activities. Fragmentsspecific to RUNX2delta8 can also used, e.g., to produce antibodies orother immune responses, as competitors or agonists, etc. These fragmentscan be referred to as being “specific for” RUNX2delta8. The latterphrase, as already defined, indicates that the peptides arecharacteristic of RUNX2delta8, and that the defined sequences aresubstantially absent from all other protein types. Such polypeptides canbe of any size which is necessary to confer specificity, e.g., 5, 8, 10,12, 15, 20, etc.

The present invention also relates to antibodies, and otherspecific-binding partners, that are specific for polypeptides encoded byRUNX2delta8. Preferred antibodies span the epitope created by thedeletion of exon 8, comprising at least amino acids SG or ISGA (SEQ IDNO: 4) at amino acid positions 333-336 of SEQ ID NO:3. Table I disclosesexamples of additional peptides that can be used to generate antibodieswhich are specific to RUNX2delta8. A core peptide comprising amino acidsISGA (SEQ ID NO: 4) at the 7/9 splice junction can comprise 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35,40, 50, etc or more amino acids at either the N- or C-terminus can beprepared to generate antibodies which specifically recognizeRUNX2delta8, without recognizing the RUNX2 form.

Antibodies can also be generated against the other RUNX isoforms. TableI provides examples of peptides that be used to create isoform specificantibodies. Suitable fragments of the peptides can also be used.

Peptides that are used to generate antibodies can contain additionalsequences at their N- or C-terminal ends that are unrelated to RUNX, butwhich are useful for coupling and to enhance immuinogenicity. Forexample, an N-terminal cysteine can be included to facilitate thepeptide's coupling to a carrier. Additional glycine residues can also beincluded in the linker region to provide flexibility to the peptide.

Antibodies, e.g., polyclonal, monoclonal, recombinant, chimeric,humanized, single-chain, Fab, and fragments thereof, can be preparedaccording to any desired method. See, also, screening recombinantimmunoglobulin libraries (e.g., Orlandi et al., Proc. Natl. Acad. Sci.,86:3833-3837, 1989; Huse et al., Science, 256:1275-1281, 1989); in vitrostimulation of lymphocyte populations; Winter and Milstein, Nature, 349:293-299, 1991. The antibodies can be IgM, IgG, subtypes, IgG2a, IgG1,etc. Antibodies, and immune responses, can also be generated byadministering naked DNA See, e.g., U.S. Pat. Nos. 5,703,055; 5,589,466;5,580,859. Antibodies can be used from any source, including, goat,rabbit, mouse, chicken (e.g., IgY; see, Duan, W0/029444 for methods ofmaking antibodies in avian hosts, and harvesting the antibodies from theeggs). An antibody specific for a polypeptide means that the antibodyrecognizes a defined sequence of amino acids within or including thepolypeptide. Other specific binding partners include, e.g., aptamers andPNA.

The preparation of polyclonal antibodies is well-known to those skilledin the art. See, for example, Green et al., Production of PolyclonalAntisera, in IMMUNOCHEMICAL PROTOCOLS (Manson, ed.), pages 1-5 (HumanaPress 1992); Coligan et al., Production of Polyclonal Antisera inRabbits, Rats, Mice and Hamsters, in CURRENT PROTOCOLS IN IMMUNOLOGY,section 2.4.1 (1992). The preparation of monoclonal antibodies likewiseis conventional. See, for example, Kohler & Milstein, Nature 256:495(1975); Coligan et al., sections 2.5.1-2.6.7; and Harlow et al.,ANTIBODIES: A LABORATORY MANUAL, page 726 (Cold Spring Harbor Pub.1988).

Antibodies can also be humanized, e.g., where they are to be usedtherapeutically. Humanized monoclonal antibodies are produced bytransferring mouse complementarity determining regions from heavy andlight variable chains of the mouse immunoglobulin into a human variabledomain, and then substituting human residues in the framework regions ofthe murine counterparts. The use of antibody components derived fromhumanized monoclonal antibodies obviates potential problems associatedwith the immunogenicity of murine constant regions. General techniquesfor cloning murine immunoglobulin variable domains are described, forexample, by Orlandi et al., Proc. Nat. Acad. Sci., 86:3833 (1989), whichis hereby incorporated in its entirety by reference. Techniques forproducing humanized monoclonal antibodies are described, for example, inU.S. Pat. No. 6,054,297, Jones et al., Nature 321: 522 (1986); Riechmannet al., Nature 332: 323 (1988); Verhoeyen et al., Science 239: 1534(1988); Carter et al., Proc. Nat'l Acad. Sci. USA 89: 4285 (1992);Sandhu, Crit. Rev. Biotech. 12: 437 (1992); and Singer et al., J.Immunol. 150: 2844 (1993).

Antibodies of the invention also may be derived from human antibodyfragments isolated from a combinatorial immunoglobulin library. See, forexample, Barbas et al., METHODS: A COMPANION TO METHODS IN ENZYMOLOGY,VOL. 2, page 119 (1991); Winter et al., Ann. Rev. Immunol. 12: 433(1994). Cloning and expression vectors that are useful for producing ahuman immunoglobulin phage library can be obtained commercially, forexample, from STRATAGENE Cloning Systems (La Jolla, Calif.).

In addition, antibodies of the present invention may be derived from ahuman monoclonal antibody. Such antibodies are obtained from transgenicmice that have been “engineered” to produce specific human antibodies inresponse to antigenic challenge. In this technique, elements of thehuman heavy and light chain loci are introduced into strains of micederived from embryonic stem cell lines that contain targeted disruptionsof the endogenous heavy and light chain loci. The transgenic mice cansynthesize human antibodies specific for human antigens and can be usedto produce human antibody-secreting hybridomas. Methods for obtaininghuman antibodies from transgenic mice are described, e.g., in Green etal., Nature Genet. 7:13 (1994); Lonberg et al., Nature 368:856 (1994);and Taylor et al., Int. Immunol. 6:579 (1994).

Antibody fragments of the present invention can be prepared byproteolytic hydrolysis of the antibody or by expression in E. coli ofnucleic acid encoding the fragment. Antibody fragments can be obtainedby pepsin or papain digestion of whole antibodies by conventionalmethods. For example, antibody fragments can be produced by enzymaticcleavage of antibodies with pepsin to provide a 5S fragment denotedF(ab′).sub.2. This fragment can be further cleaved using a thiolreducing agent, and optionally a blocking group for the sulfhydrylgroups resulting from cleavage of disulfide linkages, to produce 3.5SFab′ monovalent fragments. Alternatively, an enzymatic cleavage usingpepsin produces two monovalent Fab′ fragments and an Fc fragmentdirectly. These methods are described, for example, by Goldenberg, U.S.Pat. No. 4,036,945 and No. 4,331,647, and references contained therein.These patents are hereby incorporated in their entireties by reference.See also Nisoiihoff et al., Arch. Biochem. Biophys. 89:230 (1960);Porter, Biochem. J. 73:119 (1959); Edelman et al, METHODS IN ENZYMOLOGY,VOL. 1, page 422 (Academic Press 1967); and Coligan et al. at sections2.8.1-2.8.10 and 2.10.1-2.10.4.

Other methods of cleaving antibodies, such as separation of heavy chainsto form monovalent light-heavy chain fragments, further cleavage offragments, or other enzymatic, chemical, or genetic techniques can alsobe used. For example, Fv fragments comprise an association of V.sub.Hand V.sub.L chains. This association may be noncovalent, as described inInbar et al., Proc. Nat'l Acad. Sci. USA 69:2659 (1972). Alternatively,the variable chains can be linked by an intermolecular disulfide bond orcross-linked by chemicals such as glutaraldehyde. See, e.g., Sandhu,supra. Preferably, the Fv fragments comprise V.sub.H and V.sub.L chainsconnected by a peptide linker. These single-chain antigen bindingproteins (sFv) are prepared by constructing a structural gene comprisingnucleic acid sequences encoding the V.sub.H and V.sub.L domainsconnected by an oligonucleotide. The structural gene is inserted into anexpression vector, which is subsequently introduced into a host cellsuch as E. coli. The recombinant host cells synthesize a singlepolypeptide chain with a linker peptide bridging the two V domains.Methods for producing sFvs are described, for example, by Whitlow etal., METHODS: A COMPANION TO METHODS IN ENZYMOLOGY, VOL. 2, page 97(1991); Bird et al., Science 242:423-426 (1988); Ladneret al., U.S. Pat.No. 4,946,778; Pack et al., Bio/Technology 11: 1271-77 (1993); andSandhu, supra.

Another form of an antibody fragment is a peptide coding for a singlecomplementarity-determining region (CDR). CDR peptides (“minimalrecognition units”) can be obtained by constructing genes encoding theCDR of an antibody of interest. Such genes are prepared, for example, byusing the polymerase chain reaction to synthesize the variable regionfrom RNA of antibody-producing cells. See, for example, Larrick et al.,METHODS: A COMPANION TO METHODS IN ENZYMOLOGY, VOL. 2, page 106 (1991).

The term “antibody” as used herein includes intact molecules as well asfragments thereof, such as Fab, F(ab′)2, and Fv which are capable ofbinding to an epitopic determinant present in Bin1 polypeptide. Suchantibody fragments retain some ability to selectively bind with itsantigen or receptor. The term “epitope” refers to an antigenicdeterminant on an antigen to which the paratope of an antibody binds.Epitopic determinants usually consist of chemically active surfacegroupings of molecules such as amino acids or sugar side chains andusually have specific three dimensional structural characteristics, aswell as specific charge characteristics. Antibodies can be preparedagainst specific epitopes or polypeptide domains, such as, for example,the SG or the ISGA (SEQ ID NO:4) epitope domains of RUNX2delta8.

Antibodies which bind to RUNX2 polypeptides of the present invention canbe prepared using an intact polypeptide or fragments containing smallpeptides of interest as the immunizing antigen. See, Table 1 forexamples of peptide fragments. The polypeptide used to immunize ananimal can obtained through chemical synthesis or through recombinantmethods. The peptides can be conjugated to carriers, such as proteins.Commonly used carriers which are chemically coupled to the immunizingpeptide include keyhole limpet hemocyanin (KLH), thyroglobulin, bovineserum albumin (BSA), and tetanus toxoid.

Polyclonal or monoclonal antibodies can be further purified, forexample, by binding to and elution from a matrix to which thepolypeptide or a peptide to which the antibodies were raised is bound.Those of skill in the art will know of various techniques common in theimmunology arts for purification and/or concentration of polyclonalantibodies, as well as monoclonal antibodies (See for example, Coligan,et al., Unit 9, Current Protocols in Immunology, Wiley Interscience,1994, incorporated by reference).

Methods of Detecting Polypeptides

RUNX2 polypeptides of the present invention can be detected, visualized,determined, quantitated, etc. according to any effective method. usefulmethods include, e.g., but are not limited to, immunoassays, RIA(radioimmunassay), ELISA, (enzyme-linked-immunosorbent assay),immunoflourescence, flow cytometry, histology, electron microscopy,light microscopy, in situ assays, immunoprecipitation, Western blot,etc.

Immunoassays may be carried in liquid or on biological support. Forinstance, a sample (e.g., blood, stool, urine, cells, tissue, cerebralspinal fluid, body fluids, etc.) can be brought in contact with andimmobilized onto a solid phase support or carrier such asnitrocellulose, or other solid support that is capable of immobilizingcells, cell particles or soluble proteins. The support may then bewashed with suitable buffers followed by treatment with the detectablylabeled RUNX2delta8 specific antibody. The solid phase support can thenbe washed with a buffer a second time to remove unbound antibody. Theamount of bound label on solid support may then be detected byconventional means.

A “solid phase support or carrier” includes any support capable ofbinding an antigen, antibody, or other specific binding partner.Supports or carriers include glass, polystyrene, polypropylene,polyethylene, dextran, nylon, amylases, natural and modified celluloses,polyacrylamides, and magnetite. A support material can have anystructural or physical configuration. Thus, the support configurationmay be spherical, as in a bead, or cylindrical, as in the inside surfaceof a test tube, or the external surface of a rod. Alternatively, thesurface may be flat such as a sheet, test strip, etc. Preferred supportsinclude polystyrene beads

One of the many ways in which gene peptide-specific antibody can bedetectably labeled is by linking it to an enzyme and using it in anenzyme immunoassay (EIA). See, e.g., Voller, A., “The Enzyme LinkedImmunosorbent Assay (ELISA),” 1978, Diagnostic Horizons 2, 1-7,Microbiological Associates Quarterly Publication, Walkersville, Md.);Voller, A. et al., 1978, J. Clin. Pathol. 31, 507-520; Butler, J. E.,1981, Meth. Enzymol. 73, 482-523; Maggio, E. (ed.), 1980, EnzymeImmunoassay, CRC Press, Boca Raton, Fla. The enzyme which is bound tothe antibody will react with an appropriate substrate, preferably achromogenic substrate, in such a manner as to produce a chemical moietythat can be detected, for example, by spectrophotometric, fluorimetricor by visual means. Enzymes that can be used to detectably label theantibody include, but are not limited to, malate dehydrogenase,staphylococcal nuclease, delta-5-steroid isomerase, yeast alcoholdehydrogenase, .alpha.-glycerophosphate, dehydrogenase, triose phosphateisomerase, horseradish peroxidase, alkaline phosphatase, asparaginase,glucose oxidase, .beta.-galactosidase, ribonuclease, urease, catalase,glucose-6-phosphate dehydrogenase, glucoamylase andacetylcholinesterase. The detection can be accomplished by colorimetricmethods that employ a chromogenic substrate for the enzyme. Detectionmay also be accomplished by visual comparison of the extent of enzymaticreaction of a substrate in comparison with similarly prepared standards.

Detection may also be accomplished using any of a variety of otherimmunoassays. For example, by radioactively labeling the antibodies orantibody fragments, it is possible to detect RUNX2delta8 peptidesthrough the use of a radioimmunoassay (RIA). See, e.g., Weintraub, B.,Principles of Radioimmunoassays, Seventh Training Course on RadioligandAssay Techniques, The Endocrine Society, March, 1986. The radioactiveisotope can be detected by such means as the use of a gamma counter or ascintillation counter or by autoradiography.

It is also possible to label the antibody with a fluorescent compound.When the fluorescently labeled antibody is exposed to light of theproper wave length, its presence can then be detected due tofluorescence. Among the most commonly used fluorescent labelingcompounds are fluorescein isothiocyanate, rhodamine, phycoerythrin,phycocyanin, allophycocyanin, o-phthaldehyde and fluorescamine. Theantibody can also be detectably labeled using fluorescence emittingmetals such as those in the lanthanide series. These metals can beattached to the antibody using such metal chelating groups asdiethylenetriaminepentacetic acid (DTPA) or ethylenediaminetetraaceticacid (EDTA).

The antibody also can be detectably labeled by coupling it to achemiluminescent compound. The presence of the chemiluminescent-taggedantibody is then determined by detecting the presence of luminescencethat arises during the course of a chemical reaction. Examples of usefulchemiluminescent labeling compounds are luminol, isoluminol, theromaticacridinium ester, imidazole, acridinium salt and oxalate ester.

Likewise, a bioluminescent compound may be used to label the antibody ofthe present invention. Bioluminescence is a type of chemiluminescencefound in biological systems in which a catalytic protein increases theefficiency of the chemiluminescent reaction. The presence of abioluminescent protein is determined by detecting the presence ofluminescence. Important bioluminescent compounds for purposes oflabeling are luciferin, luciferase and aequorin.

Identifying Agents which Modulate RUNX2delta8 Activity and Expression

The present invention also relates to methods of identifying agents, andthe agents themselves, which modulate RUNX2delta8. These agents can beused to modulate the biological activity of the polypeptide encoded forthe gene, or the gene, itself. Agents which regulate the gene or itsproduct are useful in variety of different environments, including asmedicinal agents to treat or prevent disorders associated withRUNX2delta8 and as research reagents to modify the function of tissuesand cell.

Methods of identifying agents generally comprise steps in which an agentis placed in contact with the gene, transcription product, translationproduct, or other target, and then a determination is performed toassess whether the agent “modulates” the target. The specific methodutilized will depend upon a number of factors, including, e.g., thetarget (i.e., is it the gene or polypeptide encoded by it), theenvironment (e.g., in vitro or in vivo), the composition of the agent,etc.

For modulating the expression of RUNX2delta8 gene, a method cancomprise, in any effective order, one or more of the following steps,e.g., contacting a RUNX2delta88 gene (e.g., in a cell population) with atest agent under conditions effective for said test agent to modulatethe expression of RUNX2delta8, and determining whether said test agentmodulates said RUNX2 RUNX2delta8. An agent can modulate expression ofRUNX2delta8 at any level, including transcription, translation, and/orperdurance of the nucleic acid (e.g., degradation, stability, etc.) inthe cell.

For modulating the biological activity of RUNX2delta8 polypeptides, amethod can comprise, in any effective order, one or more of thefollowing steps, e.g., contacting a RUNX2delta8 polypeptide (e.g., in acell, lysate, or isolated) with a test agent under conditions effectivefor said test agent to modulate the biological activity of saidpolypeptide, and determining whether said test agent modulates saidbiological activity.

Contacting RUNX2delta8 with the test agent can be accomplished by anysuitable method and/or means that places the agent in a position tofunctionally control expression or biological activity of RUNX2delta8present in the sample. Functional control indicates that the agent canexert its physiological effect on RUNX2DELTA8 through whatever mechanismit works. The choice of the method and/or means can depend upon thenature of the agent and the condition and type of environment in whichthe RUNX2DELTA8 is presented, e.g., lysate, isolated, or in a cellpopulation (such as, in vivo, in vitro, organ explants, etc.). Forinstance, if the cell population is an in vitro cell culture, the agentcan be contacted with the cells by adding it directly into the culturemedium. If the agent cannot dissolve readily in an aqueous medium, itcan be incorporated into liposomes, or another lipophilic carrier, andthen administered to the cell culture. Contact can also be facilitatedby incorporation of agent with carriers and delivery molecules andcomplexes, by injection, by infusion, etc.

After the agent has been administered in such a way that it can gainaccess to RUNX2delta8, it can be determined whether the test agentmodulates RUNX2delta8 expression or biological activity. Modulation canbe of any type, quality, or quantity, e.g., increase, facilitate,enhance, up-regulate, stimulate, activate, amplify, augment, induce,decrease, down-regulate, diminish, lessen, reduce, etc. The modulatoryquantity can also encompass any value, e.g., 1%, 5%, 10%, 50%, 75%,1-fold, 2-fold, 5-fold, 10-fold, 100-fold, etc. To modulate RUNX2delta8expression means, e.g., that the test agent has an effect on itsexpression, e.g., to effect the amount of transcription, to effect RNAsplicing, to effect translation of the RNA into polypeptide, to effectRNA or polypeptide stability, to effect polyadenylation or otherprocessing of the RNA, to effect post-transcriptional orpost-translational processing, etc. To modulate biological activitymeans, e.g., that a functional activity of the polypeptide is changed incomparison to its normal activity in the absence of the agent. Thiseffect includes, increase, decrease, block, inhibit, enhance, etc.Biological activities of RUNX2delta8 include, e.g., ability to competewith RUNX2, ability to inhibit angiogenesis, ability to produceapoptosis of endothelial cells, and/or ability to produce apoptosis oftumor cells.

A test agent can be of any molecular composition, e.g., chemicalcompounds, biomolecules, such as polypeptides, lipids, nucleic acids(e.g., antisense to a polynucleotide sequence selected from SEQ IDNO:3), carbohydrates, antibodies, ribozymes, double-stranded RNA,aptamers, etc. For example, if a polypeptide to be modulated is acell-surface molecule, a test agent can be an antibody that specificallyrecognizes it and, e.g., causes the polypeptide to be internalized,leading to its down regulation on the surface of the cell. Such aneffect does not have to be permanent, but can require the presence ofthe antibody to continue the down-regulatory effect. Antibodies can alsobe used to modulate the biological activity a polypeptide in a lysate orother cell-free form.

Antisense RUNX2delta8 can also be used as test agents to modulate geneexpression. Antisense polynucleotide (e.g., RNA) can also be preparedfrom a polynucleotide according to the present invention, preferably ananti-sense to a sequence of SEQ ID NO:3. Antisense polynucleotide can beused in various ways, such as to regulate or modulate expression of thepolypeptides they encode, e.g., inhibit their expression, for in situhybridization, for therapeutic purposes, for making targeted mutations(in vivo, triplex, etc.) etc. For guidance on administering anddesigning anti-sense, see, e.g., U.S. Pat. Nos. 6,200,960, 6,200,807,6,197,584, 6,190,869, 6,190,661, 6,187,587, 6,168,950, 6,153,595,6,150,162, 6,133,246, 6,117,847, 6,096,722, 6,087,343, 6,040,296,6,005,095, 5,998,383, 5,994,230, 5,891,725, 5,885,970, and 5,840,708. Anantisense polynucleotides can be operably linked to an expressioncontrol sequence. A total length of about 35 by can be used in cellculture with cationic liposomes to facilitate cellular uptake, but forin vivo use, preferably shorter oligonucleotides are administered, e.g.25 nucleotides.

Antisense polynucleotides can comprise modified, normaturally-occurringnucleotides and linkages between the nucleotides (e.g., modification ofthe phosphate-sugar backbone; methyl phosphonate, phosphorothioate, orphosphorodithioate linkages; and 2′-O-methyl ribose sugar units), e.g.,to enhance in vivo or in vitro stability, to confer nuclease resistance,to modulate uptake, to modulate cellular distribution andcompartmentalization, etc. Any effective nucleotide or modification canbe used, including those already mentioned, as known in the art, etc.,e.g., disclosed in U.S. Pat. Nos. 6,133,438; 6,127,533; 6,124,445;6,121,437; 5,218,103 (e.g., nucleoside thiophosphoramidites); 4,973,679;Sproat et al., “2′-O-Methyloligoribonucleotides: synthesis andapplications,” Oligonucleotides and Analogs A Practical Approach,Eckstein (ed.), IRL Press, Oxford, 1991, 49-86; Iribarren et al.,“2′O-Alkyl Oligoribonucleotides as Antisense Probes,” Proc. Natl. Acad.Sci. USA, 1990, 87, 7747-7751; Cotton et al., “2′-O-methyl, 2′-O-ethyloligoribonucleotides and phosphorothioate oligodeoxyribonucleotides asinhibitors of the in vitro U7 snRNP-dependent mRNA processing event,”Nucl. Acids Res., 1991, 19, 2629-2635.

Methods and Agents for Modulating Endothelial Cell Proliferation andAngiogenesis

The present invention relates to methods for identifying agents whichmodulate endothelial cell proliferation and angiogenesis, methods formodulating endothelial cell proliferation and angiogenesis, and theagents themselves.

For example, the present invention relates to methods for screeningagents that modulate an endothelial cell's responsiveness to TGF-beta1,comprising one or more of the following steps, in any effectivedisorder:

a) contacting a transfected host cell with effective amounts of anagent, and optionally TGF-beta1, wherein said transfected cellcomprises: a polynucleotide comprising an expressible RUNX2delta8polynucleotide and a p21CIP1 promoter operably linked to apolynucleotide sequence coding for a detectable product; and

b) identifying whether the agent represses promoter activity as measuredby the amount measured of said detectable product.

A transfected cell is a host cell which has been engineered to expressthe RUNX2 isoform and the reporter gene. Transfection can beaccomplished routinely. An “expressible” RUNX2delta8 polynucleotidecomprises the polynucleotide sequences which enable the RUNX2delta8isoform to be transcribed into RNA and translated into polypeptide.These sequences include, e.g., promoter, enhancer, transcriptionterminator sites, etc. In addition to the RUNX2 isoform, the cell canalso be engineered to contain a p21 promoter operably linked to apolynucleotide sequence coding for a detectable product. As explained inthe examples, the p21 promoter is regulated by RUNX2. In its presence,the p21 promoter is repressed. The RUNX2delta8 isoform is able to bindto the promoter, and compete with RUNX2, but it does not substantiallyrepress it.

Methods of the present invention can be used to identify agents whichrestore the ability of the RUNX2delta8 isoform to repress p21. Thiseffect can be monitored by assaying for the appearance of the detectableproduct. The p21-detectable product construct can also be referred to asa reporter gene. In the examples, luciferase is used as the detectableproduct, but others can be used as well, e.g., beta-galactosidase, etc.Agents identified in this method can be used, e.g., to restore a cell'sability to respond to inhibition of endothelial cell proliferation byTGFbeta1. This can be used to treat diseases associated with aberrantrevascularization, such as cancer.

The present invention also relates to methods of identifying modulatorsof RUNX2delta8 in a cell population capable of forming blood vessels,comprising, one or more of the following steps in any effective order,e.g., contacting the cell population with a test agent under conditionseffective for said test agent to modulate its expression or biologicalactivity. These methods are useful, e.g., for drug discovery inidentifying and confirming the angiogenic activity of agents, foridentifying molecules in the normal pathway of angiogenesis, etc.

Any cell population capable of forming blood vessels can be utilized.Useful models, included those mentioned above, e.g., in vivoMatrigel-type assays, tumor neovascularization assays, CAM assays, BCEassays, migration assays, HUVEC growth inhibition assays, animal models(e.g., tumor growth in athymic mice), models involving hybrid cell andelectronic-based components, etc. Cells can include, e.g., endothelial,epithelial, muscle, embryonic and adult stem cells, ectodermal,mesenchymal, endodermal, neoplastic, blood, bovine CPAE (CCL-209),bovine FBHE (CRL-1395), human HUV-EC-C(CRL-1730), mouse SVEC4-10EHR1(CRL-2161), mouse MS1 (CRL-2279), mouse MS1 VEGF (CRL-2460), stem cells,etc. The phrase “capable of forming blood vessels” does not indicate aparticular cell-type, but simply that the cells in the population areable under appropriate conditions to form blood vessels. In somecircumstances, the population may be heterogeneous, comprising more thanone cell-type, only some which actually differentiate into bloodvessels, but others which are necessary to initiate, maintain, etc., theprocess of vessel formation.

The cell population can be contacted with the test agent in any mannerand under any conditions suitable for it to exert an effect on thecells, and to modulate the differentially-expressed gene or polypeptide.The means by which the test agent is delivered to the cells may dependupon the type of test agent, e.g., its chemical nature, and the natureof the cell population. Generally, a test agent must have access to thecell population, so it must be delivered in a form (or pro-form) thatthe population can experience physiologically, i.e., to put in contactwith the cells. For instance, if the intent is for the agent to enterthe cell, if necessary, it can be associated with any means thatfacilitate or enhance cell penetrance, e.g., associated with antibodiesor other reagents specific for cell-surface antigens, liposomes, lipids,chelating agents, targeting moieties, etc. Cells can also be treated,manipulated, etc., to enhance delivery, e.g., by electroporation,pressure variation, etc.

A purpose of administering or delivering the test agents to cellscapable of forming blood vessels is to determine whether they modulatethe RUNX2delta8 expression or polypeptide. By the phrase “modulate,” itis meant that the gene or polypeptide affects the polypeptide or gene insome way. Modulation includes effects on transcription, RNA splicing,RNA editing, transcript stability and turnover, translation, polypeptideactivity, and, in general, any process involved in the expression andproduction of the gene and gene product. The modulatory activity can bein any direction, and in any amount, including, up, down, enhance,increase, stimulate, activate, induce, turn on, turn off, decrease,block, inhibit, suppress, prevent, etc.

Any type of test agent can be used, comprising any material, such aschemical compounds, biomolecules, such as polypeptides (includingpolypeptide fragments and mimics), lipids, nucleic acids, carbohydrates,antibodies, small molecules, fusion proteins, etc. Test agents include,e.g., protamine (Taylor et al., Nature, 297:307, 1982), heparins,steroids, such as tetrahydrocortisol, which lack gluco- andmineral-corticoid activity (e.g., Folkman et al., Science, 221:719, 1983and U.S. Pat. Nos. 5,001,116 and 4,994,443), angiostatins (e.g., WO95/292420), triazines (e.g., U.S. Pat. No. 6,150,362), thrombospondins,endostatins, platelet factor 4, fumagillin-derivate AGH 1470,alpha-interferon, quinazolinones (e.g., U.S. Pat. No. 6,090,814),substituted dibenzothiophenes (e.g., U.S. Pat. No. 6,022,307),deoxytetracyclines, cytokines, chemokines, FGFs, antisense or siRNA toRUNX2delta8, antibodies specific for RUNX2delta8.

Whether the test agent modulates a gene or polypeptide can be determinedby any suitable method. These methods include, detecting genetranscription, detecting mRNA, detecting polypeptide and activitythereof. The detection methods includes those mentioned herein, e.g.,PCR, RT-PCR, Northern blot, ELISA, Western, RIA, yeast two-hybrid system(e.g., for identifying natural and synthetic nucleic acids and theirproducts which regulated RUNX2). In addition, further downstream targetscan be used to assess the effects of modulators, including, the presenceor absence of neoangiogenesis (e.g., using any of the mentioned testsystems, such as CAM, BCE, in vivo Matrigel-type assays) as modulated bya test agent.

The present invention also relates to methods of regulating angiogenesisin a system comprising cells, comprising administering to the system aneffective amount of a modulator of RUNX2 or RUNX2delta8, wherebyangiogenesis is regulated. A system comprising cells can be an in vivosystem, such as a heart or limb present in a patient (e.g., angiogenictherapy to treat myocardial infarction), isolated organs, tissues, orcells, in vitro assays systems (CAM, BCE, etc), animal models (e.g., invivo, subcutaneous, chronically ischemic lower limb in a rabbit model,cancer models), hosts in need of treatment (e.g., hosts suffering fromangiogenesis related diseases, such as cancer, ischemic syndromes,arterial obstructive disease, to promote collateral circulation, topromote vessel growth into bioengineered tissues, etc.

A modulator useful in such method are those mentioned already, e.g.,nucleic acid (such as an anti-sense to a gene to disrupt transcriptionor translation of the gene), antibodies (e.g., to inhibit a cell-surfaceprotein, such as an antibody specific-for the extracellular domain).Antibodies and other agents which target a polypeptide can be conjugatedto a cytotoxic or cytostatic agent, such as those mentioned already. Amodulator can also be a differentially-expressed gene, itself, e.g.,when it is desired to deliver the polypeptide to cells analogously togene therapy methods. A complete gene, or a coding sequence operablylinked to an expression control sequence (i.e., an expressible gene) canbe used to produce polypeptide in the target cells.

By the phrase “regulating angiogenesis,” it is meant that angiogenesisis effected in a desired way by the modulator. This includes,inhibiting, blocking, reducing, stimulating, inducing, etc., theformation of blood vessels. For instance, in cancer, where the growth ofnew blood vessels is undesirable, modulators of adifferentially-expressed can be used to inhibit their formation, therebytreating the cancer. Such inhibitory modulators include, e.g.,antibodies to the extracellular regions of a differentially-expressedpolypeptide, and, antisense RNA to inhibit translation of adifferentially-expressed mRNA into polypeptide (for guidance onadministering and designing anti-sense, see, e.g., U.S. Pat. Nos.6,153,595, 6,133,246, 6,117,847, 6,096,722, 6,087,343, 6,040,296,6,005,095, 5,998,383, 5,994,230, 5,891,725, 5,885,970, and 5,840,708).On the other hand, angiogenesis can be stimulated to treat ischemicsyndromes and arterial obstructive disease, to promote collateralcirculation, and to promote vessel growth into bio-engineered tissues,etc., by administering the a differentially-expressed gene orpolypeptide to a target cell population.

The present invention also relates to compositions comprisingRUNX2delta8 polypeptide or polynucleotide for treating disorders orconditions associated with neovascularization and/or excessivevascularization. As indicated in the examples below, expression ofRUNX2delta8 in endothelial cells results in apoptosis. Thus, thepolypeptide, or a polynucleotide encoding it, can be introduced intoendothelial cells to treat or prevent excessive vascularization, e.g.,to treat cancer. The polynucleotide can be introduced routinely intocells, e.g., injected directly into the tumor using naked polynucleotidewhich comprises the RUNX2delta8 coding sequence. The sequence can beinjected alone, or associated with expression control sequences. Thepolypeptide can be similarly administered by direct injection into thetarget, e.g., into a tumor. The composition can also further compriseeffective amounts of TGFbeta1, e.g., 1 microgram, 10 micrograms, 100micrograms, etc.

Reducing or knocking down the expression of RUNX2 can also be utilizedto treat cell proliferation disorders, including cancers associated withangiogenesis. Any cancer can be treated included, breast cancer. Asshown in the examples, siRNA targeting of RUNX2 can be used to treatcell proliferation disorders.

Compositions for Increasing Cell Proliferation, Survival, andAngiogenesis

The present invention also relates to compositions for increasing cellproliferation or promoting survival of stem cells, comprising: RUNX2polypeptide or polynucleotide, and/or YAP polypeptide or polynucleotide.As shown in the examples, the combination of RUNX2 and YAP (also knownas YAP65 or Yes-associated protein) can increase cell proliferation. Asexplained above, either the polynucleotide or polypeptide can beadministered. The compositions are useful in diseases or conditionswhere neo-vascularization is desired, such as heart disease. Forexample, in diseased hearts where vascular blockage is observed, thecomposition can be directly injected into the heart muscle (otherlocations) to stimulate angiogenesis. The gene or polypeptide can alsobe utilized to promote the survival of stem cells and other primary cellcultures.

Similarly, RUNX2 can also be administered for the purpose of stimulatingangiogenesis. It can be administered either as polynucleotide or aspolypeptide. Any condition that would benefit from increasevascularization can be treated, e.g., myocardial infarction, ischemicsyndromes, arterial obstructive disease, to promote collateralcirculation, to promote vessel growth into bioengineered tissues, etc.

Methods of Detecting Angiogenesis Using RUNX2delta8

The present invention also relates to detecting the presence and/orextent of blood vessels in a sample. The detected blood vessels can beestablished or pre-existing vessels, newly formed vessels, vessels inthe process of forming, or combinations thereof. A blood vessel includesany biological structure that conducts blood, including arteries, veins,capillaries, microvessels, vessel lumen, endothelial-lined sinuses, etc.These methods are useful for a variety of purposes. In cancer, forinstance, the extent of vascularization can be an important factor indetermining the clinical behavior of neoplastic cells. See, e.g.,Weidner et al., N. Engl. J. Med., 324:1-8, 1991. Thus, the presence andextent of blood vessels, including the angiogenic process itself, can beuseful for the diagnosis, prognosis, treatment, etc., of cancer andother neoplasms. Detection of vessels can also be utilized for thediagnosis, prognosis, treatment, of any diseases or conditionsassociated with vessel growth and production, to assess agents whichmodulate angiogenesis, to assess angiogenic gene therapy, etc.

An example of a method of detecting the presence or extent of bloodvessels in a sample is determining an angiogenic index of a tissue orcell sample comprising, e.g., assessing in a sample, the expressionlevels of RUNX2 or RUNX2delta8, whereby said levels are indicative ofthe angiogenic index. As shown in the Examples below, RUNX2delta8 isexpressed in tissues undergoing angiogenesis (e.g., vascular sprouting)and in endothelial cells. By the phrase “angiogenic index,” it is meantthe extent or degree of vascularity of the tissue, e.g., the number oramount of blood vessels in the sample of interest. Amounts of nucleicacid or polypeptide can be assessed (e.g., determined, detected, etc.)by any suitable method. There is no limitation on how detection isperformed.

For instance, if nucleic acid is to be assessed, e.g., an mRNAcorresponding to a differentially-expressed gene, the methods fordetecting it, assessing its presence and/or amount, can be determined byany the methods mentioned above, e.g., nucleic acid based detectionmethods, such as Northern blot analysis, RT-PCR, RACE, differentialdisplay, NASBA and other transcription based amplification systems,polynucleotide arrays, etc. If RT-PCR is employed, cDNA can be preparedfrom the mRNA extracted from a sample of interest. Once the cDNA isobtained, PCR can be employed using oligonucleotide primer pairs thatare specific for a differentially-expressed gene. The specific probescan be of a single sequence, or they can be a combination of differentsequences. A polynucleotide array can also be used to assess nucleic,e.g., where the RNA of the sample of interest is labeled (e.g., using atranscription based amplification method, such as U.S. Pat. No.5,716,785) and then hybridized to probe fixed to a solid substrate.

Polypeptide detection can also be carried out by any available method,e.g., by Western blots, ELISA, dot blot, immunoprecipitation, RIA,immunohistochemistry, etc. For instance, a tissue section can beprepared and labeled with a specific antibody (indirect or direct),visualized with a microscope, and then the number of vessels in aparticular field of view counted, where staining with antibody is usedto identify and count the vessels. Amount of a polypeptide can bequantitated without visualization, e.g., by preparing a lysate of asample of interest, and then determining by ELISA or Western the amountof polypeptide per quantity of tissue. Again, there is no limitation onhow detection is performed.

In addition to assessing the angiogenic index using an antibody orpolynucleotide probe specific for RUNX2delta8, other methods ofdetermining tissue vascularity can be applied. Tissue vascularity istypically determined by assessing the number and density of vessselspresent in a given sample. For example, microvessel density (MVD) can beestimated by counting the number of endothelial clusters in a high-powermicroscopic field, or detecting a marker specific for microvascularendothelium or other markers of growing or established blood vessels,such as CD31 (also known as platelet-endothelial cell adhesion moleculeor PECAM). A CD31 antibody can be employed in conventionalimmunohistological methods to immunostain tissue sections as describedby, e.g., Penfold et al., Br. J. Oral and Maxill. Surg., 34: 37-41; U.S.Pat. No. 6,017,949; Dellas et al., Gyn. Oncol., 67:27-33, 1997; andothers.

In addition to RUNX2delta8, other genes and their corresponding productscan be detected. For instance, it may be desired to detect a gene whichis expressed ubiquitously in the sample. A ubiquitously expressed gene,or product thereof, is present in all cell types, e.g., in about thesame amount, e.g., beta-actin. Similarly, a gene or polypeptide that isexpressed selectively in the tissue or cell of interest can be detected.A selective gene or polypeptide is characteristic of the tissue orcell-type in which it is made. This can mean that it is expressed onlyin the tissue or cell, and in no other tissue- or cell-type, or it canmean that it is expressed preferentially, differentially, and moreabundantly (e.g., at least 5-fold, 10-fold, etc., or more) when comparedto other types. The expression of the ubiquitous or selective gene orgene product can be used as a control or reference marker to compare tothe expression of differentially-expression genes. Typically, expressionof the gene can be assessed by detecting mRNA produced from it. Othermarkers for blood vessels and angiogenesis can also be detected, such asangiogenesis-related genes or polypeptides. By the phrase“angiogenesis-related,” it is meant that it is associated with bloodvessels and therefore indicative of their presence. There are a numberof different genes and gene products that are angiogenesis-related,e.g., Vezf1 (e.g., Xiang et al., Dev. Bio., 206:123-141, 1999), VEGF,VEGF receptors (such as KDR/Flk-1), angiopoietin, Tie-1 and Tie-2 (e.g.,Sato et al., Nature, 376:70-74, 1995), PECAM-1 or CD31 (e.g., DAKO,Glostrup. Denmark), CD34, factor VIII-related antigen (e.g., Brustmannet al., Gyn. Oncol., 67:20-26, 1997).

The topic headings set forth above are meant as guidance where certaininformation can be found in the application, but are not intended to bethe only source in the application where information on such topic canbe found. For other aspects of the polynucleotides, reference is made tostandard textbooks of molecular biology. See, e.g., Hames et al.,Polynucleotide Hybridization, IL Press, 1985; Davis et al., BasicMethods in Molecular Biology, Elsevir Sciences Publishing, Inc., NewYork, 1986; Sambrook et al., Molecular Cloning, CSH Press, 1989; Howe,Gene Cloning and Manipulation, Cambridge University Press, 1995; Ausubelet al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc.,1994-1998.

The entire disclosure of all applications, patents and publications,cited above and in the figures are hereby incorporated by reference intheir entirety, including U.S. Provisional Application Ser. No.60/564,979, filed Apr. 26, 2004, which is hereby incorporated byreference in its entirety.

EXAMPLES Example 1 Expression of the Runx2 Transcription Factor inAortic Vascular Sprouts

Runx2 is a member of the family of Runx/AML transcription factorscontaining a Runt DNA-binding domain, a nuclear-localization signal, andseveral activation and repression domains (Westendorf & Hiebert, 1999).Runx genes have been shown to be expressed during angiogenesis in mouseneovasculature after treatment with FGF-2 (Namba et al., 2000; Sun etal., 2001). Rat aortic tissue was used to determine whether the Runx2gene was also expressed during vascular sprouting in a physiologicallyrelevant angiogenesis assay. This model recapitulates several steps inangiogenesis, such as expansion of the EC number, formation of cell-cellcontacts, and subsequent growth arrest that are important in theformation of neovessels (Nicosia & Ottinetti, 1990). Runx2 expressionwas analyzed with Runx2-specific oligonucleotide primers correspondingto position 645 (upstream) and 1061 (downstream) in the C-terminaldomain of the mouse and human Runx2 gene (Sun et al., 2001). Theseprimers do not detect Runx1 or Runx3 expression (data not shown). Rataortic explants were cultured within fibrin gels (FIG. 1A) underconditions that induce vascular sprouting (Hiraoka et al., 1998; Nicosia& Tuszynski, 1994), expression of VEGF (Nicosia et al., 1994; Nicosia &Tuszynski, 1994), and the expression of ECM-degrading proteases such asurokinase plasminogen activator, uPA (Rabbani, 1998) and membrane-typemetalloproteinase, MT1-MMP (Hiraoka et al., 1998). A characteristic haloof vascular outgrowth was apparent 7 days after incubation of dissectedrings at 37° C. (FIG. 1A). Expression of uPA was elevated 10-fold(normalized to cyclophilin) after 7 day culture in fibrin while MT1-MMPand VEGF increased by 7 days from undetectable levels (FIG. 1B, comparelanes 1 and 3). Expression of the expected 416 bp Runx2 productincreased 3-fold (normalized to cyclophilin) in vascular sproutsrelative to freshly dissected tissue. Interestingly, another PCR product(350 bp), which was not evident in freshly dissected tissue, was highlyexpressed in vascular sprouts by day 7 (FIG. 1B, lane 3). As control,mRNA was not detectable in fibrin gel (FG) without sprouts (FIG. 1B,lane 2).

Example 2 An Alternatively Spliced Variant of RUNX2 Lacking Exon 8 isExpressed in EC

RT-PCR amplification of RUNX2 in human bone marrow EC (HBME) alsogenerated two products, one of the expected 416 bp size and a second of350 bp in size (FIG. 2A, lane 1) as shown for aortic sprouts (FIG. 1).To verify that the 416 bp and 350 bp PCR products were authentic RUNX2,bands corresponding to these products from HBME cells were sequenced(FIG. 3). Sequence comparison showed that the 416 bp product wasidentical to the expected RUNX2 mRNA, while the 350 bp product wasidentical to RUNX2 except for a deletion of 66 bp near the 3′-end of thesequence (FIG. 3A). Comparison of the sequence of the smaller productwith the published exon map (GenBank nucleotide sequence database(accession number NM_(—)004348.1/GI:10863884) and the publishedintron/exon boundaries for RUNX2 (Geoffroy et al., 1998) revealed thatthe 350 bp PCR product corresponded to a RUNX2 mRNA with exon 8 deleted(designated RUNX2Δ8). Exon 8 encodes a peptide of 22 amino acids (FIG.3B) that is unique to RUNX2 (Westendorf & Hiebert, 1999).

Previous data had shown that serum withdrawal results in rapid loss ofexpression of RUNX2 and that extracellular matrix proteins (ECM) mediateHBME differentiation, tube formation, and increased RUNX2 expression(Sun et al., 2001). The alternatively spliced RUNX2 isoform, RUNX2Δ8,also displayed a short half-life of 3 hr, relative to cyclophilincontrol in the absence of serum (FIG. 2A). Starved HBME cells wereharvested and transferred to culture plates (PL) or to culture platescoated with two different ECM substrates: either fibrin gel (FG) orMatrigel (MG) in the presence (FBS) or absence (BSA) of serum (FIG. 2B).Culture on MG increased RUNX2 and RUNX2Δ8 mRNA expression about 1.8-foldand 1.6-fold, respectively, relative to PL and was independent of thepresence of serum (FIG. 2C, compare lanes 1,2 and 3,4). However, cellscultured on a fibrin gel coating showed 1.4-fold elevated RUNX2 levelsrelative to PL only in the presence of serum (FIG. 2B, compare lanes 5and 6). In other experiments, treatment of serum-starved cells withIGF-1 as before (Sun et al., 2001) stimulated comparable increases inexpression of both RUNX2 isoforms, while RUNX2 isoforms inpost-confluent cells were undetectable (data not shown).

Example 3 RUNX2 DNA-Binding Activity and EC Proliferation

RUNX2 is a DNA-binding transcription factor that interacts with thepromoters of specific target genes (Ito, 1999; Zelzer et al., 2001). Todetermine whether RUNX2 activity was involved in EC proliferation,endogenous RUNX2 DNA-binding was measured by EMSA in proliferating(subconfluent) and growth-arrested (postconfluent) EC (FIG. 4A, 4B).Nuclear extracts from HBME cells incubated with a consensusRUNX2-binding site oligonucleotide from the osteocalcin promoter (Dueyet al., 1997) exhibited a shifted complex (FIG. 4A, lane1) that becamesuper-shifted in the presence of RUNX2-specific antibody (FIG. 4A, lanes2-5), suggesting that the predominant RUNX protein in these cells isRUNX2. Non-specific γ-tubulin antibody did not affect the shiftedcomplex (data not shown). Subconfluent HBME cells expressed more intenseDNA-binding activity than postconfluent cells (FIG. 4B, compare lanes1,2 and 5,6). The shifted complex could be competed with excess, coldoligonucleotide (FIG. 4B, lanes 3,7), but not with a non-specificoligonucleotide (FIG. 4B, lanes 4,8). Our previous data had shown thatthe angiogenic growth factor, IGF-1, increases RUNX2 mRNA and proteinlevels in HBME cells (Sun et al., 2001). Consistent with a role forRUNX2 in EC proliferation, IGF-1 treated HBME cells also exhibitedincreased RUNX2 DNA-binding activity (FIG. 4C, lane 2) relative to cellscultured in the absence of serum but not treated with IGF-1 (FIG. 4C,lane 1). IGF-1 treatment also increased expression of endogenous RUNX2and RUNX2Δ8 proteins (FIG. 4C, lane 2, panel b).

To determine whether RUNX2 or RUNX2Δ8 might regulate EC proliferation,vectors were prepared that would allow independent overexpression ofeach of these isoforms. Ectopic expression of RUNX2 and RUNX2Δ8FLAG-tagged proteins was confirmed in transiently-transfected HEK293cells (FIG. 4C, lanes 3,4). Nuclear extracts from HEK293 cellstransfected with either RUNX2 (lane 3) or RUNX2Δ8 (lane 4) expressedsimilar levels of RUNX protein (FIG. 4C, panel b) and exhibited similarRUNX2 DNA-binding activity (FIG. 4C, panel a). Pooled, stablytransfected HBME and bovine aortic EC (BAEC) also expressed ectopicRUNX2 by immunoblotting (FIG. 5A, lanes 2 and 3). Primary BAEC, whichexpress low levels of endogenous Runx2, were used in subsequent growthassays. Confluent (synchronized) BAEC transfectants were harvested,replated in serum-containing medium for 4, 24, 48, or 72 h and examinedfor growth rate using the MIT dye reduction assay. Over 3 days inculture, RUNX2 transfectants exhibited a greater increase in cell number(5.3-fold) than neo control (4.2-fold) or RUNX2Δ8 (3.6-fold)transfectants (FIG. 5B). Analysis of DNA synthesis rates, determinedusing thymidine incorporation assays, were consistent with theseresults. RUNX2 transfectants exhibited 2-fold higher thymidineincorporation by 24 and 48 hours in culture than neo or RUNX2Δ8transfectants (FIG. 5C). By 72 hr, cells had reached confluence andbecame growth arrested. In addition, treatment of serum-starvedRUNX2-transfected cells with serum for 3 or 6 hr resulted in a dramaticincrease in phosphorylated retinoblastoma (pRb) protein, indicative ofprogression into the cell cycle. pRb levels in RUNX2Δ8-transfectantsdeclined under the same conditions, while Neo transfectants showed amodest increase in pRb. These data suggest that RUNX2 is involved in theregulation of EC growth.

Example 4 TGFβ₁-Mediated Growth Inhibition and Apoptosis

TGFβ₁ functions as an angiogenic modulator by inhibiting the growth ofEC and stimulating matrix protein synthesis and EC migration (Kalluri &Sukhatme, 2000; Taipale & Keski-Oja, 1997). To determine if BAEC cellsexpressing the RUNX2 isoforms retained their sensitivity to TGFβ₁ growthmodulation, post-confluent cells were harvested, replated, and culturedin the presence or absence of TGFβ₁ for 48 hours (FIG. 6A). Treatment ofBAEC control (neo) transfectants with as little as 0.2 ng/ml TGFβ₁resulted in 50% inhibition of cell growth and 80% inhibition when cellswere treated with 20 ng/ml TGFβ₁ (FIG. 6B). RUNX2Δ8 transfectantstreated with TGFβ₁ responded similarly. However, treatment ofRUNX2-transfected cells with 0.2 or 2.0 ng/ml TGFβ₁ did not affect cellgrowth while treatment with 20 ng/ml inhibited growth by only 40%.Previous data from our laboratory had shown that expression of thedominant negative Runt DNA-binding domain of RUNX2 could inhibit ECmigration (Sun et al., 2001). To determine whether RUNX2 expressioncould regulate EC migration in the presence of TGFβ₁, BAEC transfectantswere spot cultured with a collagen overlay, treated with 10 ng/ml TGFβ,and sprouting from the central cell mass was examined (FIG. 6C, leftpanels). RUNX2-transfected cells showed a more robust sprouting responsethan NEO transfectants. RUNX2Δ8 transfected cells also exhibited asprouting response, although the cells appeared more condensed and thenuclei more fragmented than NEO or RUNX2 transfectants (FIG. 6C, rightpanels).

Cell growth is a balance of positive (proliferation) and negative(apoptosis) events and RUNX2 transfectants continued to grow pastconfluence, while RUNX2Δ8-transfected cells did not (FIG. 5) with someevidence of apoptosis when treated with TGFβ₁ (FIG. 6C). To determinewhether RUNX2Δ8 expression contributed to apoptosis, post-confluent BAECtransfectants were harvested and replated in low serum in the presenceof 2 ng/ml TGFβ₁. After 28 hours, cells were fixed with formaldehyde andstained with DAN to detect apoptotic nuclei (FIG. 7A). While neo andRUNX2 transfectants remained attached (FIG. 7B) and exhibited low levelsof apoptosis (FIG. 7C), RUNX2Δ8 transfectants appeared more looselyattached (note rounded cells in FIG. 7B) with a higher percentage ofapoptotic cells (FIG. 7C). Nuclei were condensed and in many casesfragmented, consistent with apoptosis. In addition, more detachedRUNX2Δ8 transfected cells were evident relative to neo or RUNX2transfectants (FIG. 7D) and increased cleavage of the caspase substrate,PARP, was observed in RUNX2Δ8 transfectants (FIG. 7E). By morphologicalcriteria, approximately 8-fold more RUNX2Δ8 transfectants were apoptoticthan were neo or RUNX2 transfectants (FIG. 7C). The number of RUNX2Δ8detached cells was 4 to 8-fold higher than neo or RUNX2 transfectants,respectively (FIG. 7D). However, RUNX2Δ8 transfectants did not exhibitany evidence of reduced proliferation in the absence of TGFβ₁ (FIG. 6).These results suggest that RUNX2, and more specifically its exon8domain, is involved in antagonizing TGFβ₁-mediated growth inhibition andin reducing TGFβ₁-mediated EC apoptosis.

Example 5 Regulation of the Promoter of the Cyclin-Dependent KinaseInhibitor p21^(CIP1) by RUNX2 Isoforms

Inhibition of EC proliferation by TGFβ₁ is mediated through activationof the p21^(CIP1) promoter via a p53-independent mechanism (Datto etal., 1995) whereas RUNX2 is a strong transcriptional repressor of thep21^(CIP1) promoter (Westendorf et al., 2002). To determine whetherdeletion of the exon8 domain would alter RUNX2 transcriptionalregulation of the p21^(CIP1) promoter, RUNX2Δ8 DNA binding andtransactivation functions were evaluated. EMSA using the RUNX-bindingelement in the osteocalcin promoter (FIG. 4) or a DNA precipitationassay with a biotin-labeled RUNX-consensus binding site oligonucleotidefrom the p21^(CIP1) promoter (FIG. 8A, lanes 4,6) indicated nodifferences in relative DNA binding between RUNX2 and RUNX2Δ8 isoforms.Control mutant oligonucleotide (FIG. 8A, lanes 5,7) did not interactwith RUNX2 or RUNX208. In addition, RUNX2Δ8 was able to displace RUNX2from the p21^(CIP1) promoter target oligonucleotide (FIG. 8A, lowerpanel). Therefore, the ability of RUNX2Δ8 to repress the p21^(CIP1)promoter was examined (FIG. 8 B,C,D). Co-transfection of thep21^(CIP1)-promoter-Luciferase vector with RUNX2 in NIH3T3 cellsresulted in 3-fold repression of endogenous p21^(CIP1) promoter activity(FIG. 8B), while the same dose of RUNX2Δ8 or control plasmid (neo) didnot inhibit basal activity. In cells treated with TGFβ₁ (2 ng/ml), RUNX2overexpression also repressed the p21^(CIP1) promoter (FIG. 8C) by2.2-fold, while neo and RUNX2Δ8 expression vectors did not. In thepresence of Smad3 and constitutively-active TGFβ receptor (Alk5TD),RUNX2Δ8 was able to compete with RUNX2 to inhibit RUNX2-mediatedrepression of the p21^(CIP1) promoter and to increase p21^(CIP1)promoter-Luciferase activity above baseline (FIG. 8D). Consistent withthese data, RUNX2 could also inhibit the induction of p21^(CIP1) proteinin cells treated with doxorubicin, a chemotherapeutic agent thatincreases p21^(CIP1) expression (FIG. 8D, inset). These results indicatethat the antagonism of RUNX2 for TGFβ₁-induced growth inhibition in ECmay be mediated, in part, by its ability to repress the cyclin-dependentkinase inhibitor p21^(CIP1) and that the presence of exon 8 is importantfor this effect.

Materials and Methods for Examples 1-5

Reagents

Flag-M2 monoclonal, β-tubulin, and γ-tubulin antibodies were obtainedfrom Sigma (St. Louis, Mo.). The pRb antibody, which recognizesunphosphorylated and phosphorylated forms of Rb was from BD Biosciences(cat# 554136). Human TGF-β₁ was from R&D Systems, (Minneapolis, Minn.)and the AML3/Cbfa1/RUNX2 antibody was from Oncogene Research Products(Cambridge, Mass.). Parp-specific antibody was from Roche DiagnosticsCorp. (Indianapolis, Ind.). p21^(CIP1)-specific antibody was obtainedfrom Santa Cruz, Inc. (cat# sc-397). The RUNX2 cDNA clone (PEBP2aA) wasobtained from Dr. Yoshiaki Ito (Institute of Molecular and Cell Biology,Singapore) (Ogawa et al., 1993), the p21^(CIP1)-promoter-Luciferasevector, WWP-Luc, (el-Deiry et al., 1993) from Dr. Bert Vogelstein (JohnsHopkins University, Baltimore, Md.), the Smad3 expression vector fromDrs. Mark de Caestecker (Vanderbilt University) and Anne-ChristinePoncelet (Northwestern University), and the constitutively active TGFβreceptor, Alk5TD, from Dr. Mitsuyasu Kato (University of Tsukuba,Japan).

Cell Culture

Human bone marrow EC, HBME-1, (Lehr & Pienta, 1998), a gift from Dr.Kenneth Pienta (University of Michigan Comprehensive Cancer Center) andbovine aortic endothelial cells (BAEC), purchased from the Coriell CellRepository (Camden, N.J.), were maintained in Dulbecco's modifiedEagle's medium (DMEM) containing 10% FBS and antibiotics (Penicillin,Streptomycin, AmphotericinB) from Life Technologies Inc. (Rockville,Md.). BAEC cells were used at passage 8 or less. HBME-1 were usedbetween passage 14 and 24. The human embryonic kidney 293T cells wereprovided by Dr. Robert Fenton, University of Maryland School of Medicineand cultured in DMEM containing 10% FBS.

Angiogenesis Assays

Vascular sprouting was performed using aortas from 2-month oldSprague-Dawley rats (Harlan, Indianapolis, Ind.). Tissue was dissectedand sections were implanted in fibrin gels as described (Nicosia &Ottinetti, 1990; Nicosia & Tuszynski, 1994). After culturing for oneweek at 37° C., vascular sprouts were photographed and extracted withTrizol reagent (Invitrogen, Carlsbad, Calif.) to isolate RNA. BAECsprouting in vitro was determined by plating 5×10⁴ cells in 50 ul ofcomplete medium (DMEM containing 10% FBS) for 6 hr, removing the medium,and overlaying the attached cells (“spot culture”) with a 3 mg/mlsolution of neutralized type I collagen gel (Nicosia & Ottinetti, 1990;Nicosia & Tuszynski, 1994). Complete medium was added to the cultures,cells were incubated for 3 days and sprouting from the central spots wasdocumented with a video camera attached to a Zeiss microscopy andanalyzed with image analysis software (Oncor Image, Inc.).

RT-PCR

Expression of Runx2, the protease genes uPA and MT1MMP, and theangiogenic factor, VEGF, was determined after RNA extraction usingstandard PCR methods and the following primers:5′-GCACAGACAGAAGCTTGAT-3′ (SEQ ID NO: 5) and 5′-CCCAGTTCTGAAGCACCT-3′(SEQ ID NO:6) (RUNX2/416/350 bp); 5′-CCGGACTATACAGACCATCT-3′ (SEQ ID NO:7) and 5′-AGTGTGAGACTCTCGTGTAG-3′ (SEQ ID NO: 8) (uPA/367 bp);5′-GCATTGGGTGTTTGATGAGG-3′ (SEQ ID NO:9) and 5′-GTTCTACCTTCAGCTTCTGG-3′(SEQ ID NO: 10) (MT1MMP/327 bp); 5′-TGCACCCATGGCAGAAGGAGG-3′ (SEQ ID NO:11) and 5′-TCACCGCCTCGGCTTGTCACA-3′ (SEQ ID NO: 12) (VEGF/564-360 bp);5′-CATCCTGAAGCATACAGGTC-3′ (SEQ ID NO:13) and 5′-CAGAAGGAATGGTTTGATGG-3′(SEQ ID NO: 14) (Cyclophilin/276 bp). Amplification of all genes wasperformed on a PTC-100 programmable thermal controller (MJ ResearchInc., Watertown, Mass.), as described (Sun et al., 2001). For VEGFamplification, PCR was carried out using conditions as describedpreviously (Burchardt et al., 1999). Linear conditions were establishedby varying the levels of input cDNA. Relative inducible levels ofexpression were calculated after scanning densitometry using cyclophilingene expression to normalize for loading. All PCR products weresub-cloned into the pGEM-T vector (Promega Corporation, Madison, Wis.)and sequenced at the UMB Biopolymer Core Facility to verify geneidentity.

Preparation of Expression Vectors

PCR sub-cloning with the FLAG-pCMV-Tag epitope mammalian expressionvector pCMV-Tag2 (Stratagene, La Jolla, Calif.) and the full-lengthPEBP2aA cDNA vector, pEFBoSαA1, (Ogawa et al., 1993) were used toconstruct the RUNX2 and RUNX2Δ8 expression plasmids (pCMV-Tag-RUNX2 andpCMV-Tag-RUNX2Δ8, respectively). PCR sub-cloning and the followingRUNX2-specific primers were used. Full-length RUNX2:5′-AGATCTGATGCGTATTCCTGTAGATCCG-3′ (SEQ ID NO: 15) (N-terminal with endof BglII site); 5′-CTCTCGAGTCAATATGGTCGCCAAACAG-3′ (SEQ ID NO: 16)(C-terminal with end of XhoI site). RUNX2Δ8: 5′-CAGTATGAGAGTAGGTGTCC-3′(SEQ ID NO: 17) (N-terminal; BsgI 778)(722-741);5′-AAGGGTCCACTCTGGCTTTG-3′ (SEQ ID NO: 18) (C-terminal; XcmI 1255;1248-1268). The full-length RUNX2 primers were used to amplify the fullcoding cDNA of RUNX2 with the pEF-BOSαA1 plasmid as template. Theamplified fragment was purified and subcloned into pGEM-T vector(Promega Corporation, Madison, Wis.). After sequencing to verify theRUNX2 gene, the insert was subcloned in-frame into the Bglifand XhoIsites of the pCMV-Tag epitope mammalian expression vector pCMV-Tagg(Stratagene, La Jolla, Calif.). RUNX2Δ8 expression plasmidpCMV-Tag-RUNX2Δ8 was constructed using HBME cDNA as template and PCRamplification using the RUNX2Δ8 primers above. The resulting fragment of486 base pair was subcloned into the pGEM-T vector. Sequence analysisconfirmed the identity of this short fragment to encompass the exon 8deletion. The exon 8 deletion fragment was released from pGEM-T andsubcloned into the BsgI and XcmI sites of the pCMV-Tag-RUNX2 vector.

Cell Transfection

Transfection of HBME-1 or BAEC was carried out with Superfecttransfection reagent according to the protocol provided by themanufacturer (Qiagen Inc., Valencia, Calif.) or with Lipofectin(Gibco/BRL, Rockville, Md.) or Mirus TransIT LT1 (Mirus Corporation,Madison, Wis.). Approximately Zug DNA were used for each 12 ul oftransfection reagent. Two days after transfection, stable transfectants(polyclonal) were selected with 0.8 mg/ml (HBME) or 0.4 mg/ml (BAEC) ofgeneticin (Life Technologies Inc., Rockville, Md.) for two weeks.Western blotting was used to confirm the expression of RUNX2 and RUNX2Δ8using the M2 anti-Flag-Tag antibodies as described below. Forexperimental results described in the text, four independent sets ofselected polyclonal BAEC transfectants were used after freshtransfection with the RUNX plasmids, with essentially similar resultsfrom each set. Since BAEC are primary cells with a limited life-span,experiments were carried out within two weeks of selection. HEK293 cellswere transfected using the calcium phosphate method (Gibco/BRL,Rockville, Md.).

DNA Synthesis

Thymidine incorporation into acid-insoluble DNA was determined in BAECcultured as described above. Cells were incubated for 48 h untilconfluent and sub-cultured into 96-well microliter plates. A total of1×10⁴ cells per well were seeded in triplicate. Following incubation at37° C. for 4, 8, 24, 48 or 72 h, in a humidified atmosphere with 5% CO2,³H-thymidine (1 uCi/well) was added and the incubation was continued foran additional 1 h. Cells were washed with cold Phosphate-Buffered Saline(PBS) and fixed in ice-cold 5% trichloroacetic acid to extractunincorporated radionucleotide. Acid-insoluble material from BAEC wasdissolved in 100 ul of 1N sodium hydroxide and neutralized with 100 ulof 1N HCl. ³H-thymidine incorporation was quantitated with a liquidscintillation counter (Beckman Model LS5801). Incorporation of label wasnormalized to cell protein content. Results are expressed as³H-thymidine dpm incorporated per ug protein with n=3 for eachexperimental group.

Cell Proliferation and Apoptosis Assays

BAEC cell growth was quantitated using the MTT dye reduction assay (Wang& Passaniti, 1999). Cells were cultured in 96-well plates (1×10⁴cells/well) for 4, 24, 48 or 72 hours at 37° C. in 100 ul DMEMcontaining 10% FBS. MTT dye (10 ul of a 5 mg/ml stock in PBS) was addedper well. After incubation at 37° C. for 4 hours, the crystallineformazan product was solubilized in 100 ul of 10% SDS+0.01N HCl for 16hours and absorbance at 540 nm was measured with a BioRad Model 5450plate reader. Mean and SD from the mean were calculated from n=4-6 perpoint. To measure the effect of TGFβ₁ treatment on BAEC proliferation,post-confluent cells were harvested and 1×10⁵ cells were replated perwell of a 6-well plate. Cells were cultured in growth media in thepresence or absence of TGFβ₁ (0.2, 2.0, 20 ng/ml) for 48 hours and fixedwith 3.7% PBS-buffered formaldehyde. DAPI stain (1 ug/ml; MolecularProbes, Inc., Eugene, Oreg.) was added to formaldehyde-fixed cells tovisualize nuclei and representative fields from each well werephotographed. Cell number was calculated by counting individual nucleifrom at least three fields of each well. To measure the effect of TGFβ₁on BAEC apoptosis, post-confluent cells were harvested and replated in0.2% FBS in the presence of 2 ng/ml TGFβ₁ in 6-well plates as describedfor proliferation. After 28 hours, cells were fixed with formaldehydeand stained with DAPI to detect apoptotic nuclei. Apoptosis wasquantitated by counting the number of apoptotic nuclei (usingmorphological parameters of nuclear condensation and fragmentation).Detached cells were counted by collecting the media from each well andadding 100 ul of each supernatant to glass slides, overlayed withcoverslips. To measure cleavage of the caspase 3 substrate, Parp, cellswere lysed in SDS-PAGE buffer, proteins were resolved on SDS-PAGE andthe 116 kDa/80 kDa Parp proteins were detected by Western blotting.

Protein Extraction and Immunoblot

For whole cell lysate preparation, 1×10⁷ cells were harvested and lysedin 1 ml of lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM DTT,4 mM EDTA, 50 mM NaF, 0.1 mM Na3VO₄, 1 mM PMSF, 10 ug/ml leupeptin, 10ug/ml pepstatin A, 10 ug/ml chymostatin, 1% NP-40 and 0.1% TritonX-100). After incubation at 4° C. for 15 min, the lysate was cleared bymicro-centrifugation at 10,000 g for 10 min at 4° C. The proteinconcentration in each sample was determined with the BioRad Bradfordassay. Equal amounts of protein (15 ug) were denatured by heating to 95°C. in Laemmli sample buffer and were resolved by 12% SDS-PAGE, followedby transfer to PVDF membranes. The membranes were probed with theindicated antibodies and detected using an ECL system per manufacturer'srecommendation (Amersham Pharmacia Biotech, Buckinghamshire, England).γ-tubulin was used as a loading control and was measured by re-probingthe stripped membranes with anti-γ-tubulin antibody (Sigma, St. Louis,Mo.). In some cases, immunoprecipitated protein (M2-Flag antibody) fromRIPA lysates was resolved on SDS-PAGE prior to Western blotting.

Electrophoretic Mobility Shift Assays and DNA Precipitation

Nuclear extracts were prepared by hypotonic lysis of cells andextraction of nuclear proteins with high salt buffer (Wang & Passaniti,1999). Labeled double-stranded oligonucleotide containing the consensusRUNX-binding promoter element TGTGGTT was mixed with nuclear extractsand binding complexes were resolved on 6% TBE polyacrylamide gels.Specific RUNX2 antibody (0-0.4 ug) and non-specific, control (γ-tubulin)antibody were used to verify the presence of RUNX2 in the shiftedcomplex. Cold 100-fold excess specific (RUNX binding site) or 100-foldexcess non-specific (Stat binding site) oligonucleotides were used ascontrols. For DNA precipitation assays (DNAP), wild-type or mutantdouble-stranded oligonucleotides containing a RUNX-binding site from thep21^(CIP1) promoter were incubated with nuclear extracts expressingFlag.Tag.RUNX2 or Flag.Tag.RUNX2Δ8 and specific complexes were isolatedwith streptavidin-Agarose beads.

Luciferase Assays

The Dual Luciferase kit from Promega (Madison, Wis.) was used toquantitate repression or activation of the p21-promoter by RUNX2isoforms or TGFβ, respectively. Briefly, NIH3T3 cells were plated in6-well plates at a density of 1.2×10⁵ cells/well. After 24 hrs, thecells were transfected with appropriate plasmids (neo control, RUNX2, orRUNX2Δ8, p21-promoter-luciferase, Alk5TD, Smad3) and using theTransIT-LT1 reagent (Mirus Corporation, Madison, Wis.) as suggested bythe manufacturer. The TK-Renilla vector was used to normalize luciferaseunits in each transfection. Cells were lysed 48 hrs after transfectionwith passive lysis buffer supplied in the Dual Luciferase Kit. Thelysates were then analyzed following the Dual Luciferase standardprotocol and using the Turner Design Model TD-20/20 Luminometer.

Statistical Analysis

Data was expressed as mean±S.D. Statistical differences for cell growth,DNA synthesis, apoptosis, and luciferase measurements were analyzed withStudent's t test.

Example 6

RUNX2 association with YAP and p21CIP1-promoter repression. DNA-bindingtranscription factors regulate gene expression by recruitingcorepressors or coactivators to target promoters. Yeast two-hybridscreening has shown that the WW domain of the coactivator YAP binds tothe PPxY motif (PPPYP) (SEQ ID NO: 47) of RUNX1. RUNX1 is an activatorof the osteocalcin promoter, but the addition of a YAP dominant negativeconstruct strongly inhibited osteocalcin promoter activity suggestingthat YAP is a strong transcriptional coactivator of RUNX1. Since RUNX2contains a PPxY motif that is identical to RUNX1, it was postulated thatRUNX2 also binds YAP. To show that RUNX2 and YAP interact, Flag.RUNX2and HA.YAP were expressed in HEK293 cells. Lysates were separated intocytoplasmic (C) and nuclear (N) fractions to verify the cellularlocation of each protein. Ectopic RUNX2 was localized to the nucleus,while YAP was observed in both cytoplasmic and nuclear fractions.Immunoprecipitation assays were performed on nuclear lysates with eitherthe Flag.Tag antibody or the HA.Tag antibody and the proteins wereimmunoblotted. The immunoprecipitations with the Flag.Tag antibodyidentified Flag.RUNX2 and HA.YAP in the complex, while the complementaryimmunoprecipitations with the HA.Tag antibody identified HA.YAP andRUNX2 in the complex. The immunodepleted lysates were analyzed forpossible unbound Flag.RUNX2 or HA.YAP by Western. Neither protein couldbe detected in the immunodepleted nuclear lysates. The p21CIP1 promoterwas recently identified as a target of RUNX1 repression. Ectopicexpression of RUNX2 was also shown to repress p21CIP1 promoter activityin nontransformed NIH3T3 cells. Since YAP may associate with RUNX2, wewanted to assess whether YAP could regulate repression of the p21 CIP1promoter by RUNX2. Consistent with published results, Flag.RUNX2 wasable to repress the p21 CIP1 promoter-luciferase construct in NIH3T3cells. At a constant input (0.25 ug) of NEO control or Flag.RUNX2vector, increasing amounts of HA.YAP were added to each sample. HA.YAPin the presence of NEO control had no significant effect on p21 CIP1promoter activity. As HA.YAP DNA concentration increased in the presenceof NEO, there was a slight decrease in promoter activation. However,increasing concentrations of HA.YAP alleviated the RUNX2 repression ofp21CIP1 promoter activity in a dose dependent manner, resulting incomplete relief of p21 CIP1 promoter repression at 1.0 ug DNA. Thedistal p21 CIP1 promoter contains three RUNX binding sites (RBS) nearthe p53 interacting sites. Deletion of site A resulted in almostcomplete loss of RUNX1 repression of luciferase reporter activity. Todetermine whether RUNX2 also interacted with this binding site, a 20nucleotide synthetic oligonucleotide was

created containing the consensus RBS and the flanking sequences from thedistal site A in the p21CIP1 promoter. Using DNA precipitation assays,RUNX2 was found to bind the wild type p21 CIP1 oligonucleotide but notthe mutant oligonucleotide in which the RBS had been altered.

Relief of promoter repression by YAP requires direct RUNX2 binding. Toelucidate whether a direct interaction between RUNX2 and YAP wasresponsible for the relief of RUNX2 repression of the p21CIP1 promoter,a RUNX2 mutant incapable of binding to YAP was engineered. Previousmutational analysis of the YAP binding site of RUNX1 showed that thefirst two Pro and second Tyr residues in the YAP binding domain (PPYP)(SEQ ID NO: 48) were necessary for transcriptional activation of a (tk)promoter containing a GAL4 binding site (24). Mutation of the firstproline to an alanine completely abolished transcriptional activity. Wereasoned that if YAP was directly interacting with RUNX2, a mutation ofthe first proline to an alanine in RUNX2 would reduce YAP binding andnot affect the repression of the p21 CIP1 promoter. The mutant RUNX2,designated RUNX2(P409A), was generated and its ability to interact withYAP was assessed. Co-immunopreciptiation assays with the Flag.RUNX2 orFlag.RUNX2(P409A) and HA.YAP showed that YAP could associate withwild-type RUNX2 but not with the mutant. We expected that the mutantRUNX2 would retain its ability to bind DNA since the P409A mutation wasdownstream of the Runt DNA binding domain (amino acids 50-177). Tomeasure RUNX2(P409A) DNA binding, DNA precipitation assays using thewild type 20-mer, double-stranded oligonucleotide from the p21 CIP1promoter site A were performed using nuclear lysates from HEK293 cellstransfected with two mutant Flag.RUNX2(P409A) clones. Both RUNX2(P409A)mutant clones retained DNA-binding activity. After verification of DNAbinding, the ability of the RUNX2 mutant protein to repress p21CIP1promoter activity was determined. NIH3T3 cells were transfected witheither RUNX2 or RUNX2(P409A) and luciferase activity was measured. Bothmutant RUNX2(P409A) clones and the wild-type RUNX2 were strongrepressors of the p21CIP1 promoter. However, HA.YAP was unable torelieve p21 CIP1 promoter repression by the mutant Flag.RUNX2(P409A).Since YAP relieved the RUNX2-mediated, but not RUNX2(P409A)-mediatedrepression of the p21 CIP1 promoter, these data suggest that direct YAPbinding to RUNX2 is necessary to

relieve RUNX2 repression of the p21 CIP1 promoter.

RUNX2 and YAP Synergistically Increase Oncogenic Transformation.

Overexpression of RUNX2 decreased p21CIP1 protein levels in endothelialcells which could influence cell survival. p21CIP1 interacts withspecific cyclin dependent kinases to regulate G1/S cell cycletransition. Expression of a transcriptional repressor of p21CIP1, suchas RUNX2, may alter cell cycle progression and lead to changes in cellsurvival, proliferation, and/or transformation. NIH3T3 cells weretransfected with RUNX2 or RUNX2 and YAP with the appropriate controlvectors.

Expression of RUNX2 or YAP was verified with antibodies specific for theflag or HAtag. Examination of post-confluent cultures of transfectedNIH3T3 cells revealed that RUNX2 and RUNX2+YAP-transfected cells couldgrow on top of the confluent cell monolayers and form cell foci,indicative of transformation.

Transfected cells were suspended in soft agar to measureanchorage-independent growth, another indicator of transformation. Aftertwo weeks in soft agar, the RUNX2 transfected cells formed five timesmore colonies than the NEO (vector alone) controls. Moreover, thecolonies formed by the combination of RUNX2 and YAP were two tofive-fold larger than those of RUNX2 expressing cells. The number ofcolonies formed in the presence of RUNX2+YAP was greater than the numberof colonies expected from expression of RUNX2 or YAP separately,indicating that overexpression of RUNX2 and YAP increase oncogenictransformation in a synergistic manner.

To determine whether the increased in cellular transformation was theresult of a direct interaction between RUNX2 and YAP, cells transfectedwith the mutant RUNX2(P409A) and YAP were compared in the soft agarassay. Although RUNX2(P409A) retained the ability to bind DNA andrepress the p21CIP1 promoter, it was incapable of increasing cellulartransformation in the absence of YAP and even inhibited growth in softagar below the levels of the NEO control. The coexpression of YAP andRUNX2(P409A) had no effect on transformation. These results indicate adirect interaction between RUNX2 and YAP is necessary to induce loss ofcontact inhibition and growth in soft agar, established indicators ofcellular transformation in NIH3T3 cells.

Experimental Procedures for Example 6.

Cell Culture and Reagents

NIH3T3 cells were cultured in DMEM (Biofluids) and 10% FBS (Biofluids)and used until passage 20. Stable NIH3T3 cell lines were selected in 1mg/ml G418 (Invitrogen) and only used for 3 more passages afterselection. Monoclonal anti-flag M2 antibody (Sigma) was used to detector immunoprecipitate the flag-tagged RUNX2 or RUNX2(P409A) mutantproteins. Monoclonal HA.11 antibody (Covance) was used to detect orimmunoprecipitate the HA-tagged YAP.

Plasmids and Transfections

pCMV-tag2a (NEO) was purchased from Stratagene. The fall length RUNX2cDNA was inserted into the BamHI/XhoI sites of the pCMV-tag2a aspreviously described. The YAP expression vector and the empty vectorcontrol (X540-HA) were gifts from Dr. Iain Farrance (University ofMaryland, Baltimore, Md.). The p21CIP1 promoter luciferase plasmid(WWP-LUC) was a gift from Dr. Bert Vogelstein (Johns Hopkins University,Baltimore, Md.). Using a site-directed mutanagesis kit (Invitrogen), apoint mutation was introduced into the RUNX2 cDNA, which changed theproline at position 409 to an alanine, to create the RUNX2(P409A)mutant. The mutation was verified by sequencing.

Immunoprecipitation and Western Blot Analysis

Nuclear proteins were isolated using NucBuster (Novagen). Proteinconcentration was determined with the Bio-Rad Protein Assay. 1 mg ofprotein was diluted into 500 ul of immunoprecipitation buffer (20 mMTris, pH 7.5, 2 mM CaCl2, 1% Triton X-100, and 1× protease inhibitorcocktail (Roche)) and was precleared with 30 ul of protein G sepharose.For immunoprecipitations, 1 ug of antibody (M2 or HA) pre-bound to 30 ulof Protein G sepharose and was combined with Protein G pre-clearednuclear extracts and incubated on an orbital shaker for at least 1 hr at4° C. The mixture was centrifuged, and the pellet was washed 3 timeswith the IP buffer. All excess fluid was removed and 2.5 ul reducingagent (Invitrogen) and 22.5 ul of 4× Laemmli buffer was added to thepellet. Samples were boiled for 10 min and centrifuged. The supernatantwas loaded on a 4-12% Nu-PAGE gel (Invitrogen), and electrotransferredto nitrocellulose membranes (Invitrogen). The blots were incubated witheither anti-M2 antibody (1:1000) or anti-HA antibody (1:5000) followedby horseradish peroxidase-conjugated goat anti-mouse IgG (KPL,Gaithersburg, Md.). Specific proteins were detected by enhancedchemiluminescence (ECL, Amersham Pharmcia Biotech, Buckinghamshire,England).

DNA Precipitation Assays

Two single stranded, biotin labeled oligonucleotides corresponding toRUNX binding site A in the distal p21CIP1 promoter were hybridized togenerate a double-stranded probe. For the wild-type probe, the specificoligos used were 5′GCTCAGTACCACAAAAATTC-biotin 3′ (SEQ ID NO: 19)(sense) and 5′ GAATTTTTGTGGTACTGAGC-biotin 3′ (SEQ ID NO: 20)(antisense). For the mutant probe, the specific oligos used were5′GCTCAGTCGAACAAAAATTC-biotin 3′ (SEQ ID NO: 21) (sense) and 5′GAATTTTTGTTCGACTGAGC-biotin 3′ (SEQ ID NO: 22). Equal concentrations ofsense oligo and antisense oligo were added in annealing buffer for afinal concentration of 3.33 uM of double-stranded oligo in 0.1M Tris, pH7.6, 0.01M MgCl₂, 0.0034M DTT.

The mixture was heated to 95° C. for 10 minutes, allowed to cool slowlyto 65° C., then allowed to cool to RT. Nuclear proteins were isolatedusing NucBuster (Novagen). 1 mg of protein was diluted into 500 ul ofDNAP buffer (10 mM Hepes, pH 7.0, 100 mM KCl, 5 mM MgCl2, 10% glycerol,1 mM DTT, 0.5% NP40, and 1× protease inhibitor cocktail), and sampleswere pre-cleared with 30 ul streptavidin-agarose beads (Pierce). Thebiotinylated, double stranded DNA probe (10 ul of a 3.33 uM stock) and10 ug of poly dI/dC were added to the supernatant and incubated at 4° C.overnight. To the mixture, 30 ul of the streptavidin beads were addedand the incubation continued at 4° C. for at least 1 hr. Thesupernatants were then removed and the beads were washed 3 times with0.5 ml of the DNA precipitation buffer. Laemmli buffer plus reducingagent were added to the beads and the mixture was boiled for 10 minutes.After centrifugation at 14,000 rpm for 2 min, the supernatant was loadedon a 4-12% NuPage gel to resolve proteins bound to the DNA.

Luciferase Assays

Non-transformed, early passage NIH3T3 cells were plated in 6-well platesat a density of 10⁵ cells per well. Cells were allowed to recover for 24hrs and then transfected with the indicated combination of plasmids. Forall luciferase assays, the WWP-LUC plasmid was used at a concentrationof 1 ug per well and the pTK-renilla was used at a concentration of 50ng per well. Cells were incubated at 37° C. in a 5.0% CO2 incubator for48 hrs. The cells were lysed with 1× passive lysis buffer (Promega).Lysates were analyzed using the Dual Luciferase Kit (Promega) and aTurner Design TD 20/20 luminometer.

Soft Agar Assays and Foci Formation

DMEM, 10% FBS, and agar (0.5%) mixture (2 ml) were added and allowed tocool and solidify at 25° C. for at least 30 min. Cells (20,000) in 0.5ml DMEM, 10% FBS, and agar (0.33%) were carefully overlaid on thesolidified agar base in each well. This mixture was allowed to solidifyat 25° C. for 30 minutes. The plates were then incubated for 10 days ina 37° C., 5.0% CO2. Colony formation was compared and photos ofrepresentative regions from each well were taken using a Zeissmicroscope, and video camera, and images were processed with Oncor Imagesoftware. Each photo contains multiple computer images fitted togetherto give a larger representative view of the colonies in each well. Formeasurement of foci formation, NIH3T3 cells and transfectants werecultured in 100 mm dishes and allowed to reach confluence. Cells growingabove the fibroblast monolayer were photographed 25 days afterculturing.

Example 7

To determine the ability of TGF-beta1 to inhibit HBME proliferationunder conditions where RUNX2 expression is low, HBME were pretreatedwith siRNA vectors targeting RUNX2 for 24 hours prior to treatment withTGF-beta1 (2 ng/ml) under low serum conditions (1% FBS) for anadditional 48 hours. Control treated cells (pSupNeo) exhibiteda >10-fold increase in cell numbers under these conditions whileTGF-beta1 treatment of Neo control cells resulted in only about a 3-foldincrease. Expression of siRNA alone inhibited cell proliferation (4-foldincrease in cell number) while treatment with TGF-beta1 after RUNX2knockdown inhibited proliferation even further (2-fold increase in cellnumber). Since these high levels of TGF-beta1 (2 ng/ml) may overwhelmcell proliferation even in the presence of RUNX2, the experiment wasrepeated with a lower dose of TGF-beta1 (0.1 ng/ml). Under theseconditions, TGF-beta1 inhibited proliferation more dramatically whencells were pretreated with siRNA targeting RUNX2 compared to controltreated (pSupNeo) cells.

REFERENCES

-   Alexandrow, M. G., Kawabata, M., Aakre, M. & Moses, H. L. (1995).    Proc Natl Acad Sci USA, 92, 3239-43.-   Asahara, T., Bauters, C., Zheng, L. P., Takeshita, S., Bunting, S.,    Ferrara, N., Symes, J. F. & Isner, J. M. (1995). Circulation, 92,    II365-71.-   Baltzinger, M., Mager-Heckel, A. M. & Remy, P. (1999). Dev Dyn, 216,    420-33.-   Beck, L., Jr. & D'Amore, P. A. (1997). Faseb J, 11, 365-73.-   Blyth, K., Terry, A., Mackay, N., Vaillant, F., Bell, M.,    Cameron, E. R., Neil, J. C. & Stewart, M. (2001). Oncogene, 20,    295-302.-   Bravo, J., Li, Z., Speck, N. A. & Warren, A. J. (2001). Nat Struct    Biol, 8, 371-378.-   Burchardt, M., Burchardt, T., Chen, M. W., Shabsigh, A., de la    Taille, A., Buttyan, R. & Shabsigh, R. (1999). Biol Reprod, 60,    398-404.-   Carmeliet, P. & Collen, D. (2000). J Pathol, 190, 387-405.-   Claassen, G. F. & Hann, S. R. (2000). Proc Natl Acad Sci USA, 97,    9498-503.-   Datto, M. B., Li, Y., Panus, J. F., Howe, D. J., Xiong, Y. &    Wang, X. F. (1995). Proc Natl Acad Sci USA, 92, 5545-9.-   Ducy, P., Zhang, R., Geoffroy, V., Ridall, A. L. & Karsenty, G.    (1997). Cell, 89, 747-54. el-Deiry, W. S., Tokino, T.,    Velculescu, V. E., Levy, D. B., Parsons, R., Trent, J. M., Lin, D.,    Mercer, W. E., Kinzler, K. W. & Vogelstein, B. (1993). Cell, 75,    817-25.-   Ferrara, N. (2000). Recent Prog Holm Res, 55, 15-35; discussion    35-6.-   Flaumenhaft, R., Abe, M., Mignatti, P. & Rifkin, D. B. (1992). J    Cell Biol, 118, 901-9.-   Folkman, J. (1995). Nat Med, 1, 27-31.-   Gartel, A. L. & Tyner, A. L. (1999). Exp Cell Res, 246, 280-9.-   Geoffroy, V., Corral, D. A., Zhou, L., Lee, B. & Karsenty, G.    (1998). Mamm Genome, 9, 54-7.-   Gothie, E., Richard, D. E., Berra, E., Pages, G. & Pouyssegur, J.    (2000). J Biol Chem, 275, 6922-7.-   Hanahan, D. (1997). Science, 277, 48-50.-   Hata-Sugi, N., Kawase-Kageyama, R. & Wakabayashi, T. (2002). Biol    Pharm Bull, 25, 446-51.-   Hiraoka, N., Allen, E., Apel, I. J., Gyetko, M. R. & Weiss, S. J.    (1998). Cell, 95, 365-77.-   Huang, Y. Q., Li, J. J. & Karpatkin, S. (2000). Blood, 95, 1993-9.-   Ito, Y. (1999). Genes Cells, 4, 685-96.-   Ito, Y. & Miyazono, K. (2003). Curr Opin Genet Dev, 13, 43-7.-   Jakubowiak, A., Pouponnot, C., Berguido, F., Frank, R., Mao, S.,    Massague, J. & Nimer, S. D. (2000). J Biol Chem, 275, 40282-7.-   Kalluri, R. & Sukhatme, V. P. (2000). Curr Opin Nephrol Hypertens,    9, 413-8.-   Kerbel, R. S. (2000). Carcinogenesis, 21, 505-15.-   Koff, A., Ohtsuki, M., Polyak, K., Roberts, J. M. & Massague, J.    (1993). Science, 260, 536-9.-   Komori, T., Yagi, H., Nomura, S., Yamaguchi, A., Sasaki, K.,    Deguchi, K., Shimizu, Y., Bronson, R. T., Gao, Y. H., Inada, M.,    Sato, M., Okamoto, R., Kitamura, Y., Yoshiki, S. & Kishimoto, T.    (1997). Cell, 89, 755-64.-   Kriventseva, E. V., Koch, I., Apweiler, R., Vingron, M., Bork, P.,    Gelfand, M. S. & Sunyaev, S. (2003). Trends Genet, 19, 124-8.-   Laiho, M., DeCaprio, J. A., Ludlow, J. W., Livingston, D. M. &    Massague, J. (1990). Cell, 62, 175-85.-   Lehr, J. E. & Pienta, K. J. (1998). J Natl Cancer Inst, 90, 118-23.-   Li, Q. L., Ito, K., Sakakura, C., Fukamachi, H., Inoue, K. I.,    Chi, X. Z., Lee, K. Y., Nomura, S., Lee, C. W., Han, S. B., Kim, H.    M., Kim, W. J., Yamamoto, H., Yamashita, N., Yano, T., Ikeda, T.,    Itohara, S., Inazawa, J., Abe, T., Hagiwara, A., Yamagishi, H., Ooe,    A., Kaneda, A., Sugimura, T., Ushijima, T., Bae, S. C. & Ito, Y.    (2002). Cell, 109, 113-124.-   Li, W. W. (2000). Acad Radiol, 7, 800-11.-   Linggi, B., Muller-Tidow, C., van de Locht, L., Hu, M., Nip, J.,    Serve, H., Berdel, W. E., van der Reijden, B., Quelle, D. E.,    Rowley, J. D., Cleveland, J., Jansen, J. H., Pandolfi, P. P. &    Hiebert, S. W. (2002). Nat Med, 8, 743-50.-   Lund, A. H. & Van Lohuizen, M. (2002). Cancer Cell, 1, 213-215.-   Lutterbach, B., Westendorf, J. J., Linggi, B., Isaac, S., Seto, E. &    Hiebert, S. W. (2000). J Biol Chem, 275, 651-6.-   Lyons, R. M. & Moses, H. L. (1990). Eur J Bioche™, 187, 467-73.-   Maisonpierre, P. C., Suri, C., Jones, P. F., Bartunkova, S.,    Wiegand, S. J., Radziejewski, C., Compton, D., McClain, J.,    Aldrich, T. H., Papadopoulos, N., Daly, T. J., Davis, S.,    Sato, T. N. & Yancopoulos, G. D. (1997). Science, 277, 55-60.-   Massague, J. & Wotton, D. (2000). EMBO J, 19, 1745-1754.-   Nagata, K. (1996). Trends Biochem Sci, 21, 22-6.-   Namba, K., Abe, M., Saito, S., Satake, M., Ohmoto, T., Watanabe, T.    & Sato, Y. (2000). Oncogene, 19, 106-14.-   Newton, L. K., Yung, W. K., Pettigrew, L. C. & Steck, P. A. (1990).    Exp Cell Res, 190, 127-32.-   Ngo, C. V., Gee, M., Akhtar, N., Yu, D., Volpert, 0., Auerbach, R. &    Thomas-Tikhonenko, A. (2000). Cell Growth Differ, 11, 201-10.-   Nicosia, R. F., Nicosia, S. V. & Smith, M. (1994). Am J Pathol, 145,    1023-9.-   Nicosia, R. F. & Ottinetti, A. (1990). Lab Invest, 63, 115-22.-   Nicosia, R. F. & Tuszynski, G. P. (1994). J Cell Biol, 124, 183-93.-   Nor, J. E., Christensen, J., Mooney, D. J. & Polyerini, P. J.    (1999). Am JPathol, 154, 375-84.-   Ogawa, E., Inuzuka, M., Maruyama, M., Satake, M., Naito-Fujimoto,    M., Ito, Y. & Shigesada, K. (1993). Virology, 194, 314-31.-   Otto, F., Thornell, A. P., Crompton, T., Denzel, A., Gilmour, K. C.,    Rosewell, I. R., Stamp, G. W., Beddington, R. S., Mundlos, S.,    Olsen, B. R., Selby, P. B. & Owen, M. J. (1997). Cell, 89, 765-71.-   Pepper, M. S., Belin, D., Montesano, R., Orci, L. & Vassalli, J. D.    (1990). J Cell Biol, 111, 743-55.-   Perry, C., Sklan, E. H., Birikh, K., Shapira, M., Trejo, L.,    Eldor, A. & Soreq, H. (2002). Oncogene, 21, 8428-41.-   Poliman, M. J., Naumovski, L. & Gibbons, G. H. (1999). J Cell    Physiol, 178, 359-70.-   Rabbani, S. A. (1998). In Vivo, 12, 135-42.-   Riccioni, T., Cirielli, C., Wang, X., Passaniti, A. &    Capogrossi, M. C. (1998). Gene Ther, 5, 747-54.-   Risau, W. & Flamme, I. (1995). Annu Rev Cell Dev Biol, 11, 73-91.-   Roninson, I. B. (2002). Cancer Lett, 179, 1-14.-   Sato, Y. (2000). Pharmacol Ther, 87, 51-60.-   Selvamurugan, N., Pulumati, M. R., Tyson, D. R. & Partridge, N. C.    (2000). J Biol Chem, 275, 5037-42.-   Smith, C. W. J. & Valcarcel, J. (2000). TIBS, 25, 381-388.-   Smith, J. S. (2002). Trends in Cell Biol, 12, 404-6.-   Sorek, R. & Amitai, M. (2001). Nature Biotechnol, 19, 196.-   Stewart, M., Terry, A., Hu, M., O'Hara, M., Blyth, K., Baxter, E.,    Cameron, E., Onions, D. E. & Neil, J. C. (1997). Proc Natl Acad Sci    USA, 94, 8646-51.-   Sun, L., Vitolo, M. & Passaniti, A. (2001). Cancer Res, 61,    4994-5001.-   Taipale, J. & Keski-Oja, J. (1997). Faseb J, 11, 51-9.-   Takakura, N., Watanabe, T., Suenobu, S., Yamada, Y., Noda, T., Ito,    Y., Satake, M. & Suda, T. (2000). Cell, 102, 199-209.-   Takeichi, H., Hosokawa, N., Hirayoshi, K. & Nagata, K. (1994). Mol    Cell Biol, 14, 567-575.-   Tischer, E., Mitchell, R., Hartman, T., Silva, M., Gospodarowicz,    D., Fiddes, J. C. & Abraham, J. A. (1991). J Biol Chem, 266,    11947-54.-   Vaillant, F., Blyth, K., Terry, A., Bell, M., Cameron, E. R.,    Neil, J. & Stewart, M. (1999). Oncogene, 18, 7124-34.-   Wang, W. & Passaniti, A. (1999). J Cell Biochem, 73, 321-31.-   Westendorf, J. J. & Hiebert, S. W. (1999). J Cell Biochem, 32/33,    51-8.-   Westendorf, J. J., Zaidi, S. K., Cassino, J. E., Kahler, R., van    Wijnen, A. J., Lian, J. B., Yoshida, M., Stein, G. S. & Li, X.    (2002). Mol Cell Biol, 22, 7982-92.-   Xiao, G., Jiang, D., Thomas, P., Benson, M. D., Guan, K.,    Karsenty, G. & Franceschi, R. T. (2000). J Biol Chem, 275, 4453-9.-   Xiao, Z. S., Thomas, R., Hinson, T. K. & Quarles, L. D. (1998).    Gene, 214, 187-97.-   Yang, C., Chang, J., Gorospe, M. & Passaniti, A. (1996). Cell Growth    Differ, 7, 161-71.-   Zelzer, E., Glotzer, D. J., Hartmann, C., Thomas, D., Fukai, N.,    Soker, S. & Olsen, B. R. (2001). Mech Dev, 106, 97-106.-   Zhang, Y. W., Bae, S. C., Takahashi, E. & Ito, Y. (1997). Oncogene,    15, 367-71.

TABLE I DESCRIPTION AMINO ACID SEQUENCE RUNX1 RUNX1GSIASPSVHPATPISPGRASGMTTLSAELSSRLSTAPDLTA (SEQ ID NO: 23) RUNX2 RUNX2MRIPVDPSTSRRFSPPS (SEQ ID NO: 24) Exon 8 DDDTATSDFCLWPSTLSKKSQA (SEQ IDNO: 25) Exon 8 CLWPSTLSKKSQ (SEQ ID NO: 26) Exon 8 CL S PSTLSKKSQ (SEQID NO: 27) RUNX2 QMTSPSIHSTTPLSSTRGTGLPAITDVPRRIS (SEQ ID NO: 28) RUNX2GASELGP (SEQ ID NO: 29) RUNX2Δ8 ISGA (SEQ ID NO: 4) RUNX2Δ8QMTSPSIHSTTPLSSTRGTGLPAITDVPRRISGASELGP (SEQ ID NO: 30) RUNX2Δ8 CGGGPRRISGASE (SEQ ID NO: 31) RUNX2Δ8 TDVPRRISGASELGP (SEQ ID NO: 32) RUNX3RUNX3 FDRSFPTLPTLTESRFPDPRMHYPGAMSAAFPYSATPSGT (SEQ ID NO: 33)

1. A method for reducing angiogenesis in a subject in need thereof,comprising administering to the subject a therapeutically effectiveamount of an agent that reduces the expression or biological activity ofRUNX2 polypeptide identified by amino acid SEQ ID NO: 35 or abiologically active fragment thereof, or by administering to the subjecta therapeutically effective amount of a nucleic acid encodingRUNX2delta8 polypeptide identified by amino acid SEQ ID NO: 3 or abiologically active fragment thereof, or RUNX2delta8 polypeptide or abiologically active fragment thereof.
 2. The method of claim 1, whereinthe agent is selected from the group consisting of anti-sense RNA andsiRNA that specifically binds to a gene identified by nucleotide SEQ IDNO: 34 that encodes RUNX2 polypeptide identified by amino acid SEQ IDNO: 35 or mRNA encoding the RUNX2 polypeptide thereby reducing itsexpression, or an antibody or antibody fragment that specifically bindsto the RUNX2 polypeptide or a biologically active fragment thereof,thereby reducing RUNX2 polypeptide biological activity.
 3. The method ofclaim 1, wherein the subject has a disorder or condition associated withneovascularization or excessive vascularization, or cancer or other cellproliferation disorder associated with excessive angiogenesis.
 4. Amethod of reducing angiogenesis in a system comprising cells capable offorming blood vessels, comprising contacting the system with aneffective amount of an agent that reduces the expression or biologicalactivity of RUNX2 polypeptide identified by amino acid SEQ ID NO: 35 orwith an effective amount of a nucleic acid encoding RUNX2delta8polypeptide or a biologically active fragment thereof, or byadministering to the subject a therapeutically effective amount of anucleic acid encoding RUNX2delta8 polypeptide identified by amino acidSEQ ID NO: 3 or a biologically active fragment thereof, or RUNX2delta8polypeptide or a biologically active fragment thereof.
 5. The method ofclaim 4, wherein the agent is selected from the group consisting ofanti-sense RNA and siRNA that specifically binds to a gene identified bynucleotide SEQ ID NO: 34 that encodes RUNX2 polypeptide identified byamino acid SEQ ID NO: 35 or mRNA encoding the RUNX2 polypeptideidentified by amino acid SEQ ID NO: 35, or an antibody or antibodyfragment that specifically binds to the RUNX2 polypeptide or abiologically active fragment thereof, thereby reducing RUNX2 biologicalactivity.